Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Summary

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Abstract

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

Introduction

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. In the mouse, this process begins at embryonic day (E) 9.5 with the formation of the frontonasal prominence and pairs of maxillary and mandibular processes, each of which contains post-migratory cranial neural crest cells. The lateral and medial nasal processes arise from the frontonasal prominence with the appearance of the nasal pits and eventually fuse to form the nostrils. Further, the medial nasal processes and maxillary processes fuse to generate the upper lip. Concurrently, palatogenesis is initiated with the formation of distinct outgrowths - the secondary palatal shelves - from the oral side of the maxillary processes at E11.5. Over time, the palatal shelves grow downward on either side of the tongue, elevate to an opposing position above the tongue, and eventually fuse at the midline to form a continuous palate that separates the nasal and oral cavities by E16.5¹.

These morphological changes throughout craniofacial development are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. For example, cell membrane receptors, such as subfamilies of transforming growth factor (TGF)-β receptors, including bone morphogenetic protein receptors (BMPRs), and various receptor tyrosine kinase (RTK) families, are autophosphorylated upon ligand binding and activation in cranial neural crest cells^2,3,4. Additionally, the G protein-coupled transmembrane receptor Smoothened becomes phosphorylated in cranial neural crest cells and craniofacial ectoderm downstream of Sonic hedgehog (SHH) ligand binding to the Patched1 receptor, resulting in Smoothened accumulation at the ciliary membrane and SHH pathway activation⁵. Such ligand-receptor interactions can occur through autocrine, paracrine, and/or juxtacrine signaling in craniofacial contexts. For example, BMP6 is known to signal in an autocrine manner during chondrocyte differentiation⁶, whereas fibroblast growth factor (FGF) 8 is expressed in the pharyngeal arch ectoderm and binds to members of the FGF family of RTKs expressed in the pharyngeal arch mesenchyme in a paracrine fashion to initiate patterning and outgrowth of the pharyngeal arches^7,8,9,10. Furthermore, Notch signaling is activated in both chondrocytes and osteoblasts during craniofacial skeletal development through juxtacrine signaling when transmembrane Delta and/or Jagged ligands bind to transmembrane Notch receptors on neighboring cells, which are subsequently cleaved and phosphorylated¹¹. However, there are other ligand and receptor pairs important for craniofacial development that have the flexibility to function in both autocrine and paracrine signaling. As an example, during murine tooth morphogenesis, platelet-derived growth factor (PDGF)-AA ligand has been demonstrated to signal in an autocrine manner to activate the RTK PDGFRα in the enamel organ epithelium¹². In contrast, in murine facial processes during mid-gestation, transcripts encoding the ligands PDGF-AA and PDGF-CC are expressed in the craniofacial ectoderm, while the PDGFRα receptor is expressed in the underlying cranial neural crest-derived mesenchyme, resulting in paracrine signaling^{13,14,15,16,17}. Regardless of the signaling mechanism, these receptor phosphorylation events often result in the recruitment of adaptor proteins and/or signaling molecules, which frequently become phosphorylated themselves to initiate intracellular kinase cascades such as the mitogen-activated protein kinase (MAPK) pathway^18,19.

The terminal intracellular effectors of these cascades can then phosphorylate an array of substrates, such as transcription factors, RNA-binding, cytoskeletal and extracellular matrix proteins. Runx2²⁰, Hand1²¹, Dlx3/5^22,23,24, Gli1-3²⁵, and Sox9²⁶ are among the transcription factors phosphorylated in the context of craniofacial development. This post-translational modification (PTM) can directly affect susceptibility to alternative PTMs, dimerization, stability, cleavage, and/or DNA-binding affinity, among other activities^20,21,25,26. Additionally, the RNA-binding protein Srsf3 is phosphorylated in the context of craniofacial development, leading to its nuclear translocation²⁷. In general, phosphorylation of RNA-binding proteins has been shown to affect their subcellular localization, protein-protein interactions, RNA binding, and/or sequence specificity²⁸. Furthermore, phosphorylation of actomyosin can lead to cytoskeletal rearrangements throughout craniofacial development^29,30, and phosphorylation of extracellular matrix proteins, such as small integrin-binding ligand N-linked glycoproteins, contributes to biomineralization during skeletal development³¹. Through the above and numerous other examples, it is evident that there are wide implications for protein phosphorylation during craniofacial development. Adding an additional level of regulation, protein phosphorylation is further modulated by phosphatases, which counteract kinases by removing phosphate groups.

These phosphorylation events at both the receptor and effector molecule levels are critical for the propagation of signaling pathways and ultimately result in changes in gene expression in the nucleus, driving specific cell activities, such as migration, proliferation, survival, and differentiation, which result in proper formation of the mammalian face. Given the context specificity of protein interactions with kinases and phosphatases, the resulting changes in PTMs, and their effects on cell activity, it is critical that these parameters be studied in a physiologically-relevant setting to gain complete understanding of the contribution of phosphorylation events to craniofacial development. Here, examples of two contexts in which to study phosphorylation of proteins and, thus, activation of signaling pathways during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves - both primary³² and immortalized³³. At E11.5, the maxillary processes are in the process of fusing with the lateral and medial nasal processes¹, thereby representing a critical timepoint during mouse craniofacial development. Further, maxillary processes and cells derived from the palatal shelves were chosen here because the latter structures are derivatives of the former, thereby providing researchers the opportunity to interrogate protein phosphorylation in vivo and in vitro in related contexts. However, this protocol is also applicable to alternative facial processes and developmental timepoints.

A critical problem in studying phosphorylated proteins is that they are easily dephosphorylated during protein isolation by abundant environmental phosphatases. To overcome this barrier, adaptations and modifications to standard laboratory methods that allow for isolation of phosphorylated proteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphorylated proteins. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various signaling pathways active during craniofacial development.

Protocol

All the procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus and performed in compliance with institutional guidelines and regulations. Female 129S4 mice at 1.5-6 months of age and housed at a sub-thermoneutral temperature of 21-23 °C were used for embryo harvests. A schematic workflow of the protocol is represented in Figure 1. See the Table of Materials for details regarding all materials, equipment, software, reagents, and animals used in this protocol.

1. Harvesting E11.5 mouse embryos

1. Euthanize pregnant female mouse 11.5 days after detection of vaginal plug during timed mating in a CO₂ chamber using IACUC-approved CO₂ flow rate required for approximately 50% displacement (2.95 L/min in a 360 in³ chamber) and duration of exposure (see Table of Materials). Perform cervical dislocation as a secondary method of euthanasia. Proceed immediately to dissection.
2. Lay the mouse body on a dissecting board with the ventral side facing up. Spray the mouse abdomen with 70% ethanol.
3. Open the abdominal cavity by pinching and lifting the skin anterior to the vaginal opening with straight Semken forceps and cutting the lifted skin and underlying layers with straight blade surgical scissors at a 45° angle on either side to generate a "V" shape that extends to each lateral surface roughly half-way between the forelimbs and hindlimbs.
4. Using the Semken forceps, grip one of the uterine horns and cut below the oviduct and above the cervix with the surgical scissors. Cut away the mesometrium to allow for complete removal of the uterine horn.
5. Transfer the dissected uterine horn to 10 mL of histology phosphate-buffered saline (PBS) [0.137 M NaCl, 2.68 mM KCl, 1.76 mM KH₂PO₄ monobasic, 10.14 mM Na₂HPO₄ dibasic, pH 7.4] in a 10 cm Petri dish.
6. Remove the second uterine horn on the opposing side of the abdominal cavity using the same procedure described in steps 1.4-1.5.
7. Place the 10 cm Petri dish containing both uterine horns on ice if not proceeding immediately to the dissection of individual embryos (i.e., if a second female mouse will be dissected).
8. Under a dissecting stereo microscope, carefully dissect out each embryo from the uterine horns with Dumont #5 fine forceps. Slowly pull away the myometrium, decidua, and chorion. Tear and remove the relatively transparent amnion surrounding the embryo, and sever the umbilical cord connecting the embryo to the placenta.
9. Transfer each dissected embryo to 2.5 mL of histology PBS in an individual well of a 12-well cell culture plate on ice using a cut plastic transfer pipet or an embryo spoon.
   ​NOTE: If working with a litter that may contain embryos of more than one genotype, save the amnion surrounding each embryo for genotyping by placing each in its own prelabeled 0.5 mL microcentrifuge tube on ice.

2. Dissecting maxillary processes from E11.5 mouse embryos

1. Prepare three 10 cm Petri dishes containing 10 mL of histology PBS and keep them on ice to be used in rotation between embryos.
2. Transfer one embryo from an individual well of the 12-well cell culture plate to one of the 10 cm Petri dishes with histology PBS on ice using a cut plastic transfer pipet or an embryo spoon.
3. Under the dissecting microscope, separate each maxillary process from the face using the fine forceps. First, cut the anterior side of one maxillary process along the natural indentation separating the lateral nasal process and the maxillary process (Figure 2A).
4. Second, cut the posterior side of the maxillary process along the natural indentation separating the maxillary process and the mandibular process (Figure 2A).
5. Third, to completely separate the maxillary process, make a vertical cut from the anterior to posterior sides of the maxillary process on the eye side of the maxillary process where the natural indentations referenced above end (Figure 2A-C).
6. Repeat these three cuts for the maxillary process on the opposing side of the face.
7. Using a 9" Pasteur pipet with a 2 mL small latex bulb, transfer the dissected pair of maxillary processes and a small droplet of approximately 30 µL of histology PBS to a labeled 35 mm Petri dish on ice (Figure 2D).
8. Follow steps 2.3-2.7 for each embryo, rotating the three 10 cm Petri dishes containing histology PBS on ice between embryos. Place individual pairs of dissected maxillary processes in separate 35 mm Petri dishes on ice.
   NOTE: Separation of the maxillary process ectoderm and mesenchyme (steps 2.9-2.17) can be performed after dissecting the maxillary processes of all embryos in the litter. If intact maxillary processes containing both the ectoderm and mesenchyme are desired, proceed to step 2.18 below.
9. Prepare 250 µL of fresh 2% trypsin in tissue culture PBS and 250 µL of 10% fetal bovine serum (FBS) in tissue culture PBS and store on ice.
10. Place a second, small droplet of approximately 30 µL of 2% trypsin in the 35 mm Petri dish separate from the first, small droplet of histology PBS containing the maxillary processes. Transfer the pair of maxillary processes to the small droplet of 2% trypsin using the Pasteur pipet (Figure 2D).
11. Incubate the dish on ice for 15 min.
12. Remove the 35 mm Petri dish from the ice and place under the dissecting microscope.
13. Using the fine forceps, slowly and carefully pull off the layer of ectoderm from each maxillary process (Figure 2E).
    NOTE: If the ectoderm is not separating from the mesenchyme in an intact sheet, incubate in 2% trypsin at room temperature (RT) for up to 5 additional min before continuing with separation. If the tissue starts to disintegrate in the 2% trypsin, move the maxillary processes to the 10% FBS as described below to neutralize the trypsin and finish the separation of the ectoderm and mesenchyme.
14. Once the maxillary process ectoderm and mesenchyme are separated (Figure 2F), transfer the desired tissues from the pair of maxillary processes to a third, small droplet of approximately 30 µL of 10% FBS - separate from the two previous small droplets - using the Pasteur pipet (Figure 2D).
15. Place the 35 mm Petri dish on ice to stop the trypsinization.
16. Incubate the maxillary process tissues on ice in 10% FBS for 1-2 min, and then transfer the tissues from both maxillary processes to a fourth, small droplet of approximately 30 µL of prechilled histology PBS on ice - separate from the three previous small droplets - using the Pasteur pipet (Figure 2D).
17. Incubate the maxillary process tissues in histology PBS for 1 min while gently swirling the histology PBS around the maxillary process tissues with the tip of the Pasteur pipet to ensure that all FBS is rinsed off the tissue.
18. Transfer the pair of maxillary process tissues to a labeled 1.5 mL microcentrifuge tube on ice using the Pasteur pipet, minimizing the transfer of histology PBS. Remove any excess histology PBS in the 1.5 mL microcentrifuge tube with the Pasteur pipet.
19. Follow the above steps to dissect the maxillary processes from each embryo in the litter.
20. Process the samples immediately to isolate whole cell protein lysates (below) or store at -80 °C long-term. Prior to long-term storage, snap-freeze the 1.5 mL microcentrifuge tube in a bath of 100% EtOH on dry ice for 5 min.

3. Isolating whole cell protein lysates from mouse maxillary processes

1. If maxillary processes were previously frozen at -80 °C, thaw them on ice.
2. Add 0.1 mL of ice-cold NP-40 lysis buffer (20 mM Tris HCl pH 8, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA; stored at 4 °C) with protease and phosphatase inhibitors (1x complete mini protease inhibitor cocktail [dissolved in water; stored at -20 °C], 1 mM PMSF [dissolved in isopropanol; stored at 4 °C], 10 mM NaF [stored at -20 °C; avoid freeze/thaw], 1 mM Na₃VO₄ [stored at -20 °C], 25 mM β-glycerophosphate [stored at 4 °C]) added immediately before use on ice.
   NOTE: Cell fractionation can alternatively be performed to isolate cytoplasmic, nuclear, membrane, or mitochondrial protein fractions.
3. Pipet up and down 10 times with a 200 µL pipetman.
4. Vortex for 10 s, and then pipet up and down 10 times with a 200 µL pipetman. Avoid generating bubbles.
5. Incubate at 4 °C for 2 h while rotating end over end using a 1.5 mL/2 mL paddle with a tube revolver.
6. Centrifuge the samples at 13,500 × g for 20 min at 4 °C.
7. Collect the supernatant to a new 1.5 mL microcentrifuge tube on ice with a 200 mL pipetman.
8. Quantify the protein concentration with the protein assay kit, using 10 μL of protein lysate + 10 μL of NP-40 lysis buffer for experimental samples and 3-5 dilutions of bovine serum albumin (fraction V) (BSA) in NP-40 lysis buffer at a range of 0.25-2.0 mg/mL as protein standards.
9. Proceed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (step 5) or quickly freeze the remaining lysates on dry ice and store at -80 °C long-term.

4. Isolating whole cell protein lysates from primary and/or immortalized mouse embryonic palatal mesenchyme (MEPM) cells

NOTE: Isolation and culture of primary MEPM cells from E13.5 mouse embryos and immortalized MEPM cells have been previously described^32,33,34. Stimulation of cells with growth factor (steps 4.1-4.7) can be performed prior to cell lysis. If non-stimulated cells are desired, proceed to step 4.8 below.

1. Aspirate the growth medium [Dulbecco's modified Eagle's medium (DMEM) with 50 U/mL of penicillin, 50 µg/mL of streptomycin, 2 mM L-glutamine, 10% FBS] from the MEPM cells at ~70% confluence in a 6 cm cell culture dish using a 5.75" Pasteur pipet attached to a vacuum system.
2. Wash the cells with 1 mL of tissue culture PBS.
3. Add 3 mL of serum starvation medium [DMEM with 50 U/mL of penicillin, 50 µg/mL of streptomycin, 2 mM L-glutamine, 0.1% FBS] prewarmed in a 37 °C water bath.
4. Incubate at 37 °C and 5% CO₂ for 23 h.
5. Replace the serum starvation medium with 3 mL of fresh, prewarmed serum starvation medium.
6. Incubate at 37 °C and 5% CO₂ for 1 h.
7. Stimulate the cells with the growth factor of choice at an empirically-determined concentration for the desired length of time.
8. Aspirate the medium from the MEPM cells at 80%-100% confluence using a 5.75" Pasteur pipet attached to a vacuum system.
9. Wash the cells twice with ice-cold tissue culture PBS (1 mL for a 6 cm cell culture dish); tilt the plate to the side during the last wash to ensure all the tissue culture PBS is aspirated.
10. Lyse the cells by adding ice-cold NP-40 lysis buffer with protease and phosphatase inhibitors added immediately before use (0.1 mL for a 6 cm cell culture dish) on ice.
11. Incubate the plate on ice for 5 min with rotation approximately every min to ensure complete coverage of the plate.
12. Scrape the cells off the plate using a precooled cell lifter and transfer the cell suspension to a precooled 1.5 mL microcentrifuge tube on ice.
13. Incubate the cell suspension at 4 °C for 30 min while rotating end over end using a 1.5 mL/2 mL paddle with a tube revolver.
14. Proceed with steps 3.6-3.9 described above.

5. Western blotting of whole cell protein lysates from mouse facial processes and/or MEPM cells for phosphoproteins

1. Prepare whole cell protein lysate samples for SDS-PAGE.
   1. Determine the amount of protein to be loaded, depending on protein abundance in the tissue/cell; 12.5 μg is usually sufficient for robustly-expressed proteins in whole cell protein lysates.
   2. Add an equal volume of 2x Laemmli buffer [20% glycerol, 4% SDS, 0.004% bromophenol blue, 0.125 M Tris HCl pH 6.8] with 10% β-mercaptoethanol added immediately before use.
      CAUTION: β-mercaptoethanol is skin or eye corrosive, toxic, hazardous if swallowed, and has aquatic toxicity. Handle wearing gloves, a laboratory coat, face shield, and safety glasses in a chemical fume hood. Dispose of according to Environmental Health and Safety guidelines.
   3. Mix by vortexing.
   4. Heat the samples at 100 °C for 5 min in a mini dry bath.
   5. Mix by vortexing and place the samples on ice.
   6. Centrifuge at 9,400 × g for 5 min at 4 °C.
   7. Place the samples briefly on ice before loading into SDS-PAGE gel, or store at -20 °C long-term.
2. Perform SDS-PAGE using an electrophoresis cell with a 4%-15% precast protein gel and electrophoresis buffer³⁵.
3. Electrotransfer the proteins to a polyvinylidene fluoride (PVDF) membrane using transfer buffer with 0%-20% methanol (0%-10% for proteins greater than 100 kDa; 20% for proteins less than 100 kDa) added immediately before use³⁵.
4. Block the membrane and probe for phosphoprotein of interest.
   1. Following transfer, wash the membrane in 1x tris-buffered saline (TBS) [20 mM Tris, 0.137 M NaCl, pH 7.6] for 5 min in a western blot box.
   2. Incubate the membrane in 5 mL of blocking buffer [1x TBS, 0.1% Tween 20, 5% w/v BSA; mixed well and filtered through a 25 mm syringe filter with 0.2 μm pores using a 10 mL syringe with luer tip] for 1 h in a western blot box with agitation on an orbital shaker.
      NOTE: Do not use milk as a blocking agent when characterizing phosphoproteins as it contains the phosphoprotein casein, which can cause a high, non-specific background signal.
   3. Wash the membrane three times for 5 min each in TBS-T [1x TBS, 0.1% Tween 20] in a western blot box with agitation on an orbital shaker.
   4. Incubate the membrane in 5 mL of primary antibody diluted in blocking buffer at 4 °C overnight in a 50 mL conical tube using a 50 mL paddle with a tube revolver.
      NOTE: Consult the antibody datasheet for the appropriate concentration for western blotting.
   5. Wash the membrane three times at RT for 5 min each in TBS-T in a western blot box with agitation on an orbital shaker.
   6. Incubate the membrane in 5 mL of appropriate horseradish peroxidase (HRP)-conjugated secondary antibody diluted in blocking buffer for 1 h in a western blot box with agitation on an orbital shaker.
   7. Wash the membrane three times for 5 min each in TBS-T in a western blot box with agitation on an orbital shaker.
   8. Incubate the membrane in enhanced chemiluminescence (ECL) western blotting substrate [mixing equal volumes from bottles 1 and 2; 0.5 mL of each for large (8.6 cm x 6.7 cm) membranes] for 1 min, ensuring that the entire membrane is continually exposed to the ECL western blotting substrate.
   9. Drain the membrane of excess ECL western blotting substrate and immediately develop using a chemiluminescence imager.
5. Strip the membrane and reprobe for total protein of interest.
   1. Place the PVDF membrane in a transparent pouch with polyethylene lining containing 5 mL of stripping buffer [2% SDS, 62.5 mM Tris HCl pH 6.8] with 8 μL/mL of β-mercaptoethanol added immediately before use.
   2. Incubate at 50 °C for 30 min with rocking in a hybridization oven at 11 revolutions per minute.
   3. Wash the membrane three times at RT for 5 min each in TBS-T in a western blot box with agitation on an orbital shaker.
   4. Proceed with steps 5.4.2-5.4.9 described above.
6. Quantitate western blot band densities using ImageJ software, normalizing the levels of the phosphorylated protein of interest to the levels of the total protein of interest³⁶.

Representative Results

When attempting to characterize the phosphorylation of proteins isolated from mouse facial processes and/or cultured palatal mesenchyme cells, the representative results will ideally reveal a distinct, reproducible band following western blotting with an anti-phosphoprotein antibody that runs at or near the height of the corresponding total protein band (Figure 3). However, if extensive phosphorylation of the protein occurs, there may be a slight upward shift of the phosphoprotein band compared to that of the total protein band. Further, if multiple isoforms of a protein exist in a tissue, each of which is phosphorylated, or the protein is variably subjected to additional PTMs, multiple bands may appear in the western blot with an anti-phosphoprotein antibody. If no signal is observed for the phosphoprotein of interest, despite the detection of the total protein of interest, the protein may not be phosphorylated in the chosen context, or a technical issue may have arisen with the protocol. In the latter case, western blotting of whole cell lysates can be performed with anti-phosphoserine/threonine and/or anti-phosphotyrosine antibodies to indicate whether the protocol worked as expected. As phosphorylation of proteins is abundant in craniofacial tissues^25,37, the presence of multiple phosphoprotein bands will indicate successful completion of the protocol and accurate results from the initial screening.

It is often useful to compare phosphoprotein levels between wild-type and mutant embryo tissues or in response to the treatment of cultured cells. In the former case, a difference in phosphoprotein levels between genotypes would appear as a difference in band intensities for the phosphoprotein of interest with similar levels of the total protein of interest²⁷. In the latter case, representative outcomes would reveal increased phosphoprotein band intensities compared to those of untreated cells upon treatment with, for example, a growth factor (Figure 3) and decreased band intensities following treatment with a kinase inhibitor^27,37. For quantitation of these differences, levels of the phosphoprotein of interest would be normalized to levels of the total protein of interest. The levels of the total protein of interest are expected be relatively equal between samples, a finding which should be confirmed by separate western blotting using one or more antibodies against loading and expression control proteins such as β-tubulin, β-actin, and/or GAPDH (Figure 3). For growth factor treatment of cultured cells, positive results will likely show an increase in phosphoprotein levels in a relatively short time frame of 2-15 min following stimulation (Figure 3). There should be little or no phosphorylation in the absence of growth factor treatment, unless there are other factors at play resulting in phosphorylation of the protein, examples of which include endogenous expression of growth factors by the cells, phosphorylation of the protein by another signaling molecule expressed by the cells, and/or a mutation that leads to constitutive phosphorylation of the protein in the absence of growth factor treatment. These factors should be considered in the interpretation of the results. Negative results include no change in phosphorylation in response to a timecourse of growth factor treatment, in which case the protocol should be evaluated for maintenance of phosphoproteins as detailed above. Additionally, the process of membrane stripping can occasionally affect, and reduce, total protein levels. In this case, opposite trends for the amount of the phosphoprotein of interest versus the total protein of interest would be observed. This is suboptimal, as total protein levels are expected to be relatively equal across samples, assuming equal loading and expression of the protein, with changes occurring only in phosphoprotein levels.

Figure 1: Workflow for isolation of whole cell protein lysates from mouse facial processes and cultured palatal mesenchyme cells for phosphoprotein analysis. Maxillary processes are dissected from E11.5 mouse embryos on ice, and/or the medium is aspirated from cultured MEPM cells at 80%-100% confluence, which are subsequently washed twice with ice-cold PBS. Whole cell protein lysates are isolated using lysis buffer with protease and phosphatase inhibitors, followed by SDS-PAGE, electrotransfer to a PVDF membrane, blocking of the membrane with BSA, probing for the phosphoprotein, stripping of the membrane, reprobing for total protein, and quantitation of band intensities. Abbreviations: LNP = lateral nasal process; MxP = maxillary process; MdP = mandibular process; PA2 = second pharyngeal arch; PBS = phosphate-buffered saline; FBS = fetal bovine serum; PS = palatal shelf; MEPM = mouse embryonic palatal mesenchyme; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF = polyvinylidene fluoride; p-P = phosphorylated protein. Please click here to view a larger version of this figure.

Figure 2: Dissection of maxillary processes. (A) Lateral view of an E11.5 mouse embryo with the three cuts (labeled 1-3) required to separate the MxP from the face indicated by colored lines. (B) Lateral view of an E11.5 mouse embryo following removal of MxP outlined with a dashed black line. (C) Dissected MxP. (D) A 35 mm Petri dish set-up with small droplets of PBS, 2% trypsin, and 10% FBS for optional separation of MxP ectoderm and mesenchyme. (E) MxP with Ect partially separated from Mes. (F) Separated MxP Ect and Mes. Scale bars: 1 mm (A-C,E,F), 10 mm (D). Abbreviations: LNP = lateral nasal process; MxP = maxillary process; MdP = mandibular process; PA2 = second pharyngeal arch; PBS = phosphate-buffered saline; FBS = fetal bovine serum; Mes = mesenchyme; Ect = ectoderm. Please click here to view a larger version of this figure.

Figure 3: PDGFRβ and MAPK effector Erk1/2 phosphorylation in response to PDGF-BB ligand treatment of immortalized MEPM cells. Western blot analysis of whole cell lysates from immortalized MEPM cells following a timecourse of 10 ng/mL PDGF-BB ligand stimulation from 2 min to 15 min with anti-phospho-PDGFR, anti-PDGFRβ, anti-phospho-Erk1/2, anti-Erk1/2, and anti-β-tubulin (E7) antibodies. Multiple bands in anti-PDGFRβ blot represent differentially glycosylated proteins. Abbreviations: PDGF = platelet-derived growth factor; WCL = whole cell lysate; kD = kilodalton; WB = western blot; p-PDGFR = phosphorylated PDGFR; PDGFR = PDGF receptor; p-Erk = phosphorylated Erk; Erk = extracellular signal-regulated kinase. Please click here to view a larger version of this figure.

Discussion

The protocol described here allows researchers to probe critical phosphorylation-dependent signaling events during craniofacial development in a robust and reproducible manner. There are several critical steps in this protocol that ensure proper collection of data and analysis of results. Whether isolating phosphoproteins from mouse facial processes and/or cultured palatal mesenchyme cells, it is imperative to move quickly and efficiently while keeping all reagents and materials on ice when indicated. The low temperature of ice slows down metabolic activity in the cells, thereby protecting phosphorylated proteins from phosphatase activity³⁸ and maintaining a high protein yield. The use of three separate 10 cm Petri dishes during the dissection of maxillary processes ensures that all dissections take place in sufficiently prechilled histology PBS. Relatedly, an additional, essential component of this protein isolation protocol is the use of phosphatase inhibitors in all lysis buffers. These reagents also protect phosphate groups on the protein of interest^39,40 and maintain the integrity of the phosphorylated protein for further analysis by western blotting.

This protocol additionally introduces modifications that allow for more specific analyses of phosphoproteins in tissue and cultured cells. In the process of dissecting facial processes, optional steps have been added for the separation of the maxillary process ectoderm and mesenchyme to allow for the study of compartment-specific protein phosphorylation²⁷. To test for efficient isolation of these tissue layers, users can assess the expression of genes enriched in the ectoderm, such as Gabrp, Trim29, Esrp1, and Mpzl2, and/or genes enriched in the mesenchyme, such as Aldh1a2 and AW551984⁴¹. For the isolation of phosphoproteins from cultured palatal mesenchyme cells, optional steps have been added for growth factor stimulation. In this case, cells should be serum starved in medium containing 0.0%-0.1% serum. Standard serums such as FBS contain numerous growth factors⁴² that can convolute results from exogenous growth factor stimulation. Thus, temporary serum starvation primes cells to respond to acute growth factor treatment.

This protocol also provides optimized methods to accurately quantify protein phosphorylation. In the western blotting steps, it is important to block the PVDF membrane using BSA rather than milk, which is used in many standard western blotting techniques. This switch is imperative, as milk contains the phosphoprotein casein⁴³ and leads to high background signal in the analysis of other phosphoproteins. Further, it is important that levels of the phosphorylated protein of interest are quantified against levels of the total protein of interest rather than levels of a standard loading control protein. This way, any changes in the expression of the total protein of interest can be accounted for in the quantification of phosphoprotein levels. If protein yield in general is too low for accurate phosphoprotein detection, tissue samples from the same stage and genotype may be pooled²⁷, and/or cells can be grown in larger cell culture vessels than those described here for future experiments. Alternatively, if stripping consistently appears to alter the total protein levels, two approaches can be taken to modify the described protocol to achieve reproducible results: 1) use of an antibody scheme that employs anti-phosphoprotein and anti-protein antibodies generated in different host species combined with two species-specific secondary antibodies and separate (if using HRP-conjugated secondary antibodies) or simultaneous (if using fluorophore-conjugated secondary antibodies) development of the blots; or 2) simultaneous processing of two identical membranes - the first incubated with an anti-phosphoprotein antibody, the second with an anti-protein antibody. In both alternative approaches, the levels of the phosphorylated protein of interest should still be quantified against the levels of the total protein of interest. Finally, as phosphorylation is a dynamic process, phosphoprotein levels should be assessed in at least three biological replicates per condition. Mouse facial process tissue or primary MEPM cells derived from an individual embryo or an individual pool of embryos from the same stage and genotype would constitute a single biological replicate. Similarly, immortalized MEPM cells of different passages would constitute biological replicates. If cultured cells are treated with growth factor, treatment of biological replicates should occur on different days using separate aliquots of growth factor.

Two limitations of the approach presented here exist, which are discussed below and should be considered before initiating the protocol. The first involves the dynamic range of ECL. The ECL western blotting substrate recommended in this protocol has a wide dynamic range, with a low picogram sensitivity. However, empirically, proteins of low abundance or low phosphorylation status may be difficult to detect using these methods. If phosphorylated proteins are undetectable following 20 min of ECL substrate incubation, it is recommended to wash the membrane in TBS-T as described and instead incubate the membrane in a high-sensitivity ECL western blotting substrate with a low femtogram sensitivity. Alternatively, fluorophore-conjugated secondary antibodies can be used, which offer a wider dynamic range than enzyme-based approaches such as ECL⁴⁴. A second limitation arises from the normalization procedure. As described extensively, it is recommended that signal from the phosphoprotein of interest be normalized to the signal from the total protein of interest. However, this process relies on the assumption that the levels of the total protein of interest do not change between samples, which is often confirmed by separate western blotting using antibodies against one or more housekeeping proteins. An important consideration though is that the levels of housekeeping proteins may change between samples due to uneven loading, incomplete transfer to the membrane, and/or differences in protein expression. Thus, to enhance accuracy during normalization, researchers can consider moving to implement a true total protein stain, such as Ponceau S⁴⁵, which detects all proteins in each lane of the membrane and accounts for these technical limitations.

Overall, this protocol overcomes the problem of protein dephosphorylation that commonly occurs during protein isolation. Accurate analysis of protein phosphorylation will help researchers gain a more precise understanding of the role of phosphorylated proteins during craniofacial development. Furthermore, this protocol offers robust and efficient methods to analyze phosphorylated protein levels in physiologically-relevant contexts, each with their own benefits. While protein lysates from embryo tissues provide a static snapshot of protein phosphorylation in vivo, the implementation of cultured cells supplies data on dynamic phosphorylation responses to acute treatments. This protocol has the capacity to directly confirm and expand upon results and hypotheses stemming from large datasets, e.g., mass spectrometry³⁷ and RNA-sequencing²⁷, to discover new interactions within signaling networks. Importantly, this protocol can be used to probe how perturbations in phosphorylation directly influence intracellular signaling, gene expression, and cellular activity in craniofacial contexts. These diverse applications will greatly enhance understanding of the effects of protein phosphorylation in craniofacial development.

Materials

Name	Company	Catalog Number	Comments
Equipment
Block for mini dry bath	Research Products International Corp	400783
ChemiDoc XRS+ imaging system with Image Lab software	Bio-Rad	1708265	chemiluminescence imager
CO2 incubator, air jacket	VWR	10810-902
Dissecting board, 11 x 13 in	Fisher Scientific	09 002 12
Electrophoresis cell, 4-gel, for mini precast gels with mini trans-blot module	Bio-Rad	1658030
Hybridization oven	Fisher Scientific	UVP95003001
Microcentrifuge 5415 D with F45-24-11 rotor (Eppendorf)	Sigma Aldrich	Z604062
Mini dry bath	Research Products International Corp	400780
Orbital shaker	VWR	89032-092
pH meter	VWR	89231-662
Power supply for SDS-PAGE	Bio-Rad	1645050
Rectangular ice pan, maxi 9 L	Fisher Scientific	07-210-093
Stemi 508 stereo microscope with stand K LAB, LED ring light	Zeiss	4350649020000000	dissecting microscope
Timer	VWR	62344-641
Tube revolver	Fisher Scientific	11 676 341
Vortex mixer	Fisher Scientific	02 215 414
Water bath	VWR	89501-472
Western blot box	Fisher Scientific	NC9358182
Materials
Cell culture dishes, 6 cm	Fisher Scientific	12-565-95
Cell culture plates, 12 well	Fisher Scientific	07-200-82
Cell lifters	Fisher Scientific	08-100-240
CO2	Airgas	CD USP50
Conical tubes, polypropylene, 50 mL	Fisher Scientific	05-539-13
Dumont #5 fine forceps	Fine Science Tools	11254-20
Embryo spoon	Fine Science Tools	10370-17
Microcentrifuge tubes, 0.5 mL	VWR	89000-010
Microcentrifuge tubes, 1.5 mL	VWR	20170-038
Pasteur pipet, 5.75"	Fisher Scientific	13-678-6A
Pasteur pipet, 9"	VWR	14672-380
Petri dishes, 10 cm	Fisher Scientific	08-757-100D
Petri dishes, 35 mm	Fisher Scientific	FB0875711YZ
Pouches, transparent, polyethylene lining	Fisher Scientific	01-812-25B
PVDF membrane	Fisher Scientific	IPVH00010
Semken forceps	Fine Science Tools	11008-13
Small latex bulb, 2 mL	VWR	82024-554
Surgical scissors	Fine Science Tools	14002-12
Syringe filter, 25 mm, 0.2 μm pore size	Fisher Scientific	09-740-108
Syringe with luer tip, 10 mL	VWR	BD309604
Transfer pipet	Fisher Scientific	13-711-22
Western blot cassette opening lever	Bio-Rad	4560000
Whatmann 3MM chr chromatography paper	Fisher Scientific	05-714-5
Reagents
4-15% Precast protein gels, 10-well, 30 µL	Bio-Rad	4561083
β-glycerophosphate disodium salt hydrate	Sigma Aldrich	G5422-25G	stock concentration 1 M
β-mercaptoethanol	Sigma Aldrich	M3148-100ML
Bovine serum albumin, fraction V, heat shock tested	Fisher Scientific	BP1600-100
Bromophenol blue	Fisher Scientific	AC403140050
Complete mini protease inhibitor cocktail	Sigma Aldrich	11836153001	stock concentration 25x
DC protein assay kit II	Bio-Rad	500-0112
DMEM, high glucose	Gibco	11965092
E7, mouse monoclonal beta tubulin primary antibody, concentrate 0.1 mL	Developmental Studies Hybridoma Bank	E7	1:1,000
ECL western blotting substrate	Fisher Scientific	PI32106	low picogram range
ECL western blotting substrate	Genesee Scientific	20-302B	low femtogram range
Electrophoresis buffer, 5 L	Bio-Rad	1610772	stock concentration 10x
Ethanol, 200 proof, 1 gallon	Decon Laboratories, Inc.	2705HC	EtOH
Ethylenediaminetetraacetic acid, Di Na salt dihydr. (crystalline powd./electrophor.)	Fisher Scientific	BP120-500	EDTA
Fetal bovine serum, characterized, US origin, 500 mL	HyClone	SH30071.03
Glycerol (certified ACS)	Fisher Scientific	G33-4
HRP-conjugated secondary antibody, goat anti-mouse IgG	Jackson ImmunoResearch Laboratories	115-035-146	1:20,000
HRP-conjugated secondary antibody, goat anti-rabbit IgG	Jackson ImmunoResearch Laboratories	111-035-003	1:20,000
Hydrochloric acid solution, 6N (certified)	Fisher Scientific	SA56-500	HCl
Igepal Ca - 630 non-ionic detergent	Fisher Scientific	ICN19859650	Nonidet P-40
Isopropanol (HPLC)	Fisher Scientific	A451-1
L-glutamine	Gibco	25030081	stock concentration 200 mM
Methanol	Fisher Scientific	A454-4
p44/42 MAPK (Erk1/2) primary antibody	Cell Signaling Technology	9102S	1:1,000; anti-Erk1/2
PDGF-BB recombinant ligand, rat	Fisher Scientific	520BB050
PDGF Receptor β primary antibody	Cell Signaling Technology	3169S	1:1,000
Penicillin-Streptomycin	Gibco	15140122	stock concentration 100 U/mL, 100 µg/mL
Phenylmethanesulfonyl fluoride, 99%	Fisher Scientific	AC215740100	PMSF; stock concentration 100 mM
Phospho-p44/42 MAPK (Erk1/2) primary antibody	Cell Signaling Technology	9101S	1:1,000, anti-phospho-Erk1/2
Phospho-PDGF Receptor α /PDGF Receptor β primary antibody	Cell Signaling Technology	3170S	1:1,000
Potassium chloride (white crystals)	Fisher Scientific	BP366-500	KCl
Potassium phosphate monobasic (white crystals)	Fisher Scientific	BP362-500	KH2PO4
SDS solution, 10%	Bio-Rad	161-0416
Sodium chloride (crystalline/biological,certified)	Fisher Scientific	S671-3	NaCl
Sodium fluoride (powder/certified ACS)	Fisher Scientific	S299-100	NaF; aliquot for one time use; stock concentration 1 M
Sodium orthovanadate, 99%	Fisher Scientific	AC205330500	Na3VO4; stock concentration 100 mM
Sodium phosphate dibasic anhydrous (granular or powder/certified ACS)	Fisher Scientific	S374-500	Na2HPO4
Tissue culture PBS	Fisher Scientific	21-031-CV
Transfer buffer, 5 L	Bio-Rad	1610771	stock concentration 10x
Tris base (white crystals or crystalline powder/molecular biology)	Fisher Scientific	BP152-1
Trypsin	BioWorld	21560033
Tween 20	Fisher Scientific	BP337-500
Western blot molecular weight marker	Bio-Rad	1610374
Software
ImageJ software	National Institutes of Health
Animals
Female 129S4 mice			gift of Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai