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Method Article

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

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DOI:

10.3791/63834

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April 1st, 2022

 ,  , 
1Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus

* These authors contributed equally

In This Article

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 cells2,3,4. 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 arches7,8,9,10. 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 signaling13,14,15,16,17. 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) pathway18,19.

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, Dlx3/522,23,24, 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 activities20,21,25,26. 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 development29,30, 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.

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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 described32,33,34. 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.

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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 compa...

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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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

129S4 mice were a gift from Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai. This work was supported with funds from the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) R01 DE027689 and K02 DE028572 to K.A.F., F31 DE029976 to M.A.R. and F31 DE029364 to B.J.C.D.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Equipment
Block for mini dry bathResearch Products International Corp400783
ChemiDoc XRS+ imaging system with Image Lab softwareBio-Rad1708265chemiluminescence imager
CO2 incubator, air jacketVWR10810-902
Dissecting board, 11 x 13 inFisher Scientific09 002 12
Electrophoresis cell, 4-gel, for mini precast gels with mini trans-blot moduleBio-Rad1658030
Hybridization ovenFisher ScientificUVP95003001
Microcentrifuge 5415 D with F45-24-11 rotor (Eppendorf)Sigma AldrichZ604062
Mini dry bathResearch Products International Corp400780
Orbital shakerVWR89032-092
pH meterVWR89231-662
Power supply for SDS-PAGEBio-Rad1645050
Rectangular ice pan, maxi 9 LFisher Scientific07-210-093
Stemi 508 stereo microscope with stand K LAB, LED ring lightZeiss4350649020000000dissecting microscope
TimerVWR62344-641
Tube revolverFisher Scientific11 676 341
Vortex mixerFisher Scientific02 215 414
Water bathVWR89501-472
Western blot boxFisher ScientificNC9358182
Materials
Cell culture dishes, 6 cmFisher Scientific12-565-95
Cell culture plates, 12 wellFisher Scientific07-200-82
Cell liftersFisher Scientific08-100-240
CO2AirgasCD USP50
Conical tubes, polypropylene, 50 mLFisher Scientific05-539-13
Dumont #5 fine forcepsFine Science Tools11254-20
Embryo spoonFine Science Tools10370-17
Microcentrifuge tubes, 0.5 mLVWR89000-010
Microcentrifuge tubes, 1.5 mLVWR20170-038
Pasteur pipet, 5.75"Fisher Scientific13-678-6A
Pasteur pipet, 9"VWR14672-380
Petri dishes, 10 cmFisher Scientific08-757-100D
Petri dishes, 35 mmFisher ScientificFB0875711YZ
Pouches, transparent, polyethylene liningFisher Scientific01-812-25B
PVDF membraneFisher ScientificIPVH00010
Semken forcepsFine Science Tools11008-13
Small latex bulb, 2 mLVWR82024-554
Surgical scissorsFine Science Tools14002-12
Syringe filter, 25 mm, 0.2 μm pore sizeFisher Scientific09-740-108
Syringe with luer tip, 10 mLVWRBD309604
Transfer pipetFisher Scientific13-711-22
Western blot cassette opening leverBio-Rad4560000
Whatmann 3MM chr chromatography paperFisher Scientific05-714-5
Reagents
4-15% Precast protein gels, 10-well, 30 µLBio-Rad4561083
β-glycerophosphate disodium salt hydrateSigma AldrichG5422-25Gstock concentration 1 M
β-mercaptoethanolSigma AldrichM3148-100ML
Bovine serum albumin, fraction V, heat shock testedFisher ScientificBP1600-100
Bromophenol blueFisher ScientificAC403140050
Complete mini protease inhibitor cocktailSigma Aldrich11836153001stock concentration 25x
DC protein assay kit IIBio-Rad500-0112
DMEM, high glucoseGibco11965092
E7, mouse monoclonal beta tubulin primary antibody, concentrate 0.1 mLDevelopmental Studies Hybridoma BankE71:1,000
ECL western blotting substrateFisher ScientificPI32106low picogram range
ECL western blotting substrateGenesee Scientific20-302Blow femtogram range
Electrophoresis buffer, 5 LBio-Rad1610772stock concentration 10x
Ethanol, 200 proof, 1 gallonDecon Laboratories, Inc.2705HCEtOH
Ethylenediaminetetraacetic acid, Di Na salt dihydr. (crystalline powd./electrophor.)Fisher ScientificBP120-500EDTA
Fetal bovine serum, characterized, US origin, 500 mLHyCloneSH30071.03
Glycerol (certified ACS)Fisher ScientificG33-4
HRP-conjugated secondary antibody, goat anti-mouse IgGJackson ImmunoResearch Laboratories115-035-1461:20,000
HRP-conjugated secondary antibody, goat anti-rabbit IgGJackson ImmunoResearch Laboratories111-035-0031:20,000
Hydrochloric acid solution, 6N (certified)Fisher ScientificSA56-500HCl
Igepal Ca - 630 non-ionic detergentFisher ScientificICN19859650Nonidet P-40
Isopropanol (HPLC)Fisher ScientificA451-1
L-glutamineGibco25030081stock concentration 200 mM
MethanolFisher ScientificA454-4
p44/42 MAPK (Erk1/2) primary antibodyCell Signaling Technology9102S1:1,000; anti-Erk1/2
PDGF-BB recombinant ligand, ratFisher Scientific520BB050
PDGF Receptor β primary antibodyCell Signaling Technology3169S1:1,000
Penicillin-StreptomycinGibco15140122stock concentration 100 U/mL, 100 µg/mL
Phenylmethanesulfonyl fluoride, 99%Fisher ScientificAC215740100PMSF; stock concentration 100 mM
Phospho-p44/42 MAPK (Erk1/2) primary antibodyCell Signaling Technology9101S1:1,000, anti-phospho-Erk1/2
Phospho-PDGF Receptor α /PDGF Receptor β primary antibodyCell Signaling Technology3170S1:1,000
Potassium chloride (white crystals)Fisher ScientificBP366-500KCl
Potassium phosphate monobasic (white crystals)Fisher ScientificBP362-500KH2PO4
SDS solution, 10%Bio-Rad161-0416
Sodium chloride (crystalline/biological,certified)Fisher ScientificS671-3NaCl
Sodium fluoride (powder/certified ACS)Fisher ScientificS299-100NaF; aliquot for one time use; stock concentration 1 M
Sodium orthovanadate, 99%Fisher ScientificAC205330500Na3VO4; stock concentration 100 mM
Sodium phosphate dibasic anhydrous (granular or powder/certified ACS)Fisher ScientificS374-500Na2HPO4
Tissue culture PBSFisher Scientific21-031-CV
Transfer buffer, 5 LBio-Rad1610771stock concentration 10x
Tris base (white crystals or crystalline powder/molecular biology)Fisher ScientificBP152-1
TrypsinBioWorld21560033
Tween 20Fisher ScientificBP337-500
Western blot molecular weight markerBio-Rad1610374
Software
ImageJ softwareNational Institutes of Health
Animals
Female 129S4 micegift of Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai

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Protein Lysate IsolationCraniofacial DevelopmentProtein PhosphorylationWestern BlotPhosphatase InhibitorsCell Lysis BufferSignal Transduction
Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis
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