Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

摘要

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

摘要

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson's disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N',N'-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

引言

PD is one of the most common neurodegenerative diseases, predominantly affecting dopaminergic neurons in the midbrain, resulting in dysfunction of the motor systems in elderly people¹. While most patients develop PD in a sporadic manner, there are families inheriting the disease. Mutations in several genes have been found to be linked with FPD². One of the causative genes for FPD is LRRK2, in which eight missense mutations (N1437H, R1441C/G/H/S, Y1699C, G2019S, and I2020T) linked to a dominantly inherited FPD called PARK8 have so far been reported^3,4,5. Several genome-wide association studies (GWAS) of sporadic PD patients have also identified genomic variations at the LRRK2 locus as a risk factor for PD, suggesting that abnormality in the function of LRRK2 is a common cause of neurodegeneration in both sporadic and PARK8 FPD^6,7,8.

LRRK2 is a large protein (2,527 amino acids) consisting of a leucine-rich repeat domain, a GTP-binding Ras of complex proteins (ROC) domain, a C-terminal of ROC (COR) domain, a serine/threonine protein kinase domain, and a WD40 repeat domain⁹. The eight FPD mutations locate in these functional domains; N1437H and R1441C/G/H/S in the ROC domain, Y1699C in the COR domain, G2019S and I2020T in the kinase domain. Since G2019S mutation, which is the most frequently found mutation in PD patients^10,11,12, increases the kinase activity of LRRK2 by 2 - 3 fold in vitro¹³, it is hypothesized that the abnormal increase in phosphorylation of LRRK2 substrate(s) is toxic to neurons. However, it has been impossible to study whether the phosphorylation of physiologically relevant LRRK2 substrates is altered in familial/sporadic PD patients due to the lack of methods evaluating it in patient derived samples.

Protein phosphorylation is generally detected by immunoblotting or enzyme-linked immunosorbent assay (ELISA) using antibodies specifically recognizing the phosphorylated state of proteins or by mass spectrometric analysis. However, the former strategy sometimes cannot be applied because of the difficulties in creating phosphorylation-specific antibodies. Metabolic labeling of cells with radioactive phosphate is another option to examine physiological levels of phosphorylation when phosphorylation-specific antibodies are not readily available. However, it requires a large amount of radioactive materials and therefore involves some specialized equipment for radioprotection¹⁴. Mass spectrometric analysis is more sensitive compared to these immunochemical methods and became popular in analyzing protein phosphorylation. However, the sample preparation is time-consuming, and expensive instruments are required for the analysis.

A subset of the Rab GTPase family including Rab10 and Rab8 was recently reported as direct physiological substrates for LRRK2 based on the result of a large-scale phosphoproteomic analysis¹⁵. We then demonstrated that Rab10 phosphorylation was increased by FPD mutations in mouse embryonic fibroblasts and in the lungs of knockin mice¹⁶. In this report, we chose to employ a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-based method in which a P-tag molecule is co-polymerized into SDS-PAGE gels (P-tag SDS-PAGE) for detecting the endogenous levels of Rab10 phosphorylation, because a highly sensitive antibody specific for phosphorylated Rab10 was still lacking. We have failed to detect the phosphorylation of endogenous Rab8 due to the poor selectivity of currently available antibodies for total Rab8. Therefore, we decided to focus on the Rab10 phosphorylation. LRRK2 phosphorylates Rab10 at Thr73 locating at the middle of the highly conserved "switch II" region. High conservation of the phosphorylation sites among Rab proteins might be one of the reasons why phosphospecific antibodies recognizing distinct Rab proteins are difficult to make.

The phosphorylation of Rab8A by LRRK2 inhibits the binding of Rabin8, a guanine nucleotide exchange factor (GEF) which activates Rab8A by exchanging the bound GDP with GTP¹⁵. Phosphorylation of Rab10 and Rab8A by LRRK2 also inhibits the binding of GDP-dissociation inhibitors (GDIs), which are essential to the activation of Rab proteins by extracting GDP-bound Rab proteins from membranes¹⁵. Collectively, it is hypothesized that the phosphorylation of Rab proteins by LRRK2 prevents them from activation although the precise molecular mechanism and physiological consequences of the phosphorylation remain unclear.

P-tag SDS-PAGE was invented by Kinoshita et al. in 2006: In this method, acrylamide was covalently coupled with P-tag, a molecule capturing phosphates with high affinity, which copolymerized into SDS-PAGE gels¹⁷. Because the P-tag molecules in a SDS-PAGE gel selectively retard electrophoretic mobility of phosphorylated proteins, P-tag SDS-PAGE can separate phosphorylated proteins from non-phosphorylated ones (Figure 1). If the protein-of-interest is phosphorylated on multiple residues, a ladder of bands corresponding to differentially phosphorylated forms will be observed. In the case of Rab10, we observe only one shifted band, indicating that Rab10 is phosphorylated only at Thr73. The major advantage of P-tag SDS-PAGE over immunoblotting with phosphorylation-specific antibodies is that phosphorylated Rab10 can be detected by immunoblotting with non-phosphorylation-specific antibodies (i.e., recognizing total Rab10) after being transferred on membranes, which is generally more specific, sensitive, and available from commercial/academic sources. Another advantage of using P-tag SDS-PAGE is that one can obtain approximate estimation of the stoichiometry of phosphorylation, which is impossible by immunoblotting with phosphorylation-specific antibodies or by metabolic labeling of cells with radioactive phosphates.

Apart from the use of inexpensive P-tag acrylamide and some minor modifications related to it, the present method for detection of Rab10 phosphorylation by LRRK2 follows a general protocol of immunoblotting. Therefore, it should be straightforward and easily executable in any laboratories where immunoblotting is a usual practice, with any types of samples including purified proteins, cell lysates, and tissue homogenates.

方案

1. Sample Preparation for the P-tag SDS–PAGE

1. Remove and discard the media from 10 cm dishes in which cells are grown using a suction and wash cells with 5 mL Dulbecco's phosphate-buffered saline (DPBS) by first adding DPBS to the side of the dishes to avoid disturbing the cell layer, and manually rock the dishes back and forth several times.
2. Remove and discard the DPBS using a suction and add 2 mL of 0.25% (w/v) trypsin diluted in DPBS, and gently rock the dishes to cover the cell layer. Put the dishes into a CO₂ incubator (37 °C, humidified air, 5% CO₂) for 5 min.
3. After pipetting up and down using a disposable pipette to break up detached cells, collect the cell suspension into a 15 mL tube and measure the cell density using a hematocytometer under a microscope¹⁸.
4. Dilute the cells to 2.5 × 10⁵ cells/mL with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Add 2 mL (5 × 10⁵ cells) of the diluted cell suspension to each well of 6-well plates.
5. Grow the cells overnight in a CO₂ incubator (37 °C, humidified air, 5% CO₂).
6. Transfection into HEK293 cells
   NOTE: If phosphorylation of endogenous Rab10 is to be examined, proceed to step 1.7. Plasmids used in this protocol can be obtained from the authors upon request. See Table of Materials for brief information and Figure S1 for their DNA sequences.
   1. Aliquot 200 µL of DMEM into 1.5 mL tubes.
   2. To each tube, add both 0.266 µg (final concentration of 1.33 µg/mL) of a plasmid encoding HA-Rab10 and 1.066 µg (final concentration of 5.33 µg/mL) of a plasmid encoding 3×FLAG-LRRK2 from 500 µg/mL plasmid stocks. Then add 4 µL of 1 mg/mL solution of polyethyleneimine (dissolved in 20 mM HEPES-NaOH, pH 7.0) and immediately mix the solution by vortexing for 5 s.
   3. Let the tubes stand at room temperature for 10 min and add the content of one tube dropwise to one well using a micropipette. Gently and manually rock the culture plates back and forth several times to let the transfection mixture diffuse evenly throughout the well.
7. Let the cells grow for another 24-36 h in a CO₂ incubator (37 °C, humidified air, 5% CO₂).
8. If phosphorylation of endogenous Rab10 is to be examined, treat cells with and without LRRK2 inhibitors for 1 h before lysing cells.
   1. Prepare stock solutions of LRRK2 inhibitors by dissolving the inhibitors in dimethyl sulfoxide (DMSO) at 10 mM. We recommend using MLi-2 and GSK2578215A, which are highly specific and potent LRRK2 inhibitors. Store stock solutions at -80 °C.
   2. Prepare working stocks of the inhibitors by diluting the stock solutions with DMSO: For MLi-2, prepare 10 µM and 30 µM for endogenous and overexpressed LRRK2, respectively. For GSK2578215A, prepare 1 mM and 3 mM for endogenous and overexpressed LRRK2, respectively.
   3. Add 2 µL of the working stocks of either MLi-2 or GSK2578215A to the middle of one well using a micropipette and gently rock the plate manually back and forth several times to let the inhibitors diffuse evenly throughout the well.
   4. Add 2 µL of DMSO to a different well as a negative control in a similar manner to the inhibitor in step 1.8.3.
   5. Put the plates back to the incubator and culture the cells for 1 h.
9. Lyse the cells.
   1. Put the culture plates on ice. Remove and discard the media. Wash the cells by first adding 2 mL DPBS to the side of the dishes to avoid disturbing the cell layer and manually rock the dishes back and forth several times.
   2. Remove and discard the DPBS, and add to the cells 100 µL of the lysis buffer (50 mM Tris-HCl pH 7.5, 1% (v/v) polyoxyethylene(10) octylphenyl ether, 1 mM EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid), 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 0.1 µg/mL microcystin-LR, 270 mM sucrose, protease inhibitor cocktail).
   3. Tilt the plates on ice and scrape the cells using a cell scraper to gather as much cell lysate as possible. Collect the lysates using a micropipette into 1.5 mL tubes (pre-chilled on ice).
      Caution: Microcystin-LR can be fatal if swallowed or in contact with skin.
10. Let the tubes stand on ice for 10 min for complete lysis. Clarify the cell lysates by centrifugation (20,000 × g, 10 min at 4 °C) and transfer the supernatants to new 1.5 mL tubes pre-chilled on ice.
11. Measure protein concentration (µg/µL) of the cleared lysates by Bradford assay.
    1. Prepare bovine serum albumin (BSA) standards (0.2, 0.4, 0.6, 0.8, and 1 mg/mL) by diluting the stock solution with distilled water. Dilute the cleared cell lysates by 20-fold with distilled water.
    2. Put 5 µL/well of the BSA standards, blank (distilled water), and each diluted cell lysate into a 96-well plate in triplicate.
    3. Add 150 µL/well of the Bradford assay reagent using 12-channel micropipette and let the plate stand at room temperature for 5 min.
    4. Measure the absorbance at 595 nm on a plate reader and compare to BSA standards.
12. Prepare 100 µL of samples for SDS-PAGE. The protein concentration of the samples is 1 µg/µL for overexpressed HA-Rab10 and 2 µg/µL for endogenous Rab10.
    1. Using the quantified concentrations from step 1.11.4, calculate the volume (µL) of the cell lysates equivalent to 100 µg (overexpressed HA-Rab10) or 200 µg (endogenous Rab10) by dividing the protein amount (100 µg or 200 µg) by the protein concentration of lysates (µg/µL).
    2. Add 25 µL of 4× Laemmli's SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol, 0.02% (w/v) bromophenol blue, 4% (v/v) β-mercaptoethanol) to new 1.5 mL tubes kept at room temperature.
    3. Add the calculated volume of the cell lysates to each tube and mix by vortexing for 5 s at room temperature.
    4. Bring the total volume to 100 µL with the lysis buffer and mix by vortexing for 5 s at room temperature.
13. Supplement with 10 mM MnCl₂ to quench the chelating agent. Add 1 µL of 500 mM MnCl₂. Mix by vortexing for 5 s at room temperature.
    NOTE: Samples containing MnCl₂ may not be suitable for normal SDS-PAGE. This step is required due to the presence of the chelating agent (such as ethylenediaminetetraacetic acid (EDTA), EGTA, etc.) in the lysis buffer. Otherwise, Mn²⁺ ions coupled to P-tag acrylamide will be dissociated by the chelating agents, and P-tag SDS-PAGE will not work properly.
14. Boil all samples at 100 °C for 5 min and store the samples below -20 °C until use. The boiled samples can be stored up to at least 6 months.

2. Casting Gels for P-tag SDS–PAGE

NOTE: Gels should be made on the same day as running the gels. Gels can be made under ambient light conditions.

1. Prepare 5 mM P-tag acrylamide stock solution by first dissolving the 10 mg of powder/solid P-tag acrylamide completely with 100 µL of methanol and then bring to 3.3 mL by adding double distilled water.
   NOTE: P-tag acrylamide is light sensitive. The prepared solution should be stored in the dark at 4 °C until use.
2. Clean both plain and notched plates by spraying 70% ethanol and wiping with a paper towel. Assemble the gel plates. The dimensions of the gel plates used in this particular protocol are 80 or 100 mm long by 100 mm wide. Clean silicon spacers are put in between plain and notched plates and the assembled plates are clamped with binder clips.
   NOTE: Any type of gel plates that work for ordinary SDS-PAGE can be used.
3. Put a comb to be used (17-well plastic comb) into the assembled gel plates and mark on the gel plate the position of the bottom of the wells with a permanent marker.
4. Prepare 10 mL of 10% acrylamide gel mixture (10% (w/v) acrylamide (acrylamide:bis-acrylamide = 29:1), 375 mM Tris-HCl (pH 8.8), 0.1% (w/v) SDS) in a 15 mL tube.
   NOTE: The optimal concentration of acrylamide may vary depending on the reagents used. Tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) should not be added at this point.
5. Add 100 µL of 5 mM P-tag acrylamide and 10 µL of 1 M MnCl₂ solutions at final concentrations of 50 µM and 100 µM, respectively.
   NOTE: The optimal concentrations of the P-tag acrylamide and MnCl₂ might also vary depending on the reagents used.
6. Add 15 µL of TEMED to the gel mixture at a final concentration of 0.15% (v/v) and then 50 µL of 10% (w/v) APS at a final concentration of 0.05% (w/v). Mix well by gently swirling the tube for 5 s and pour into the assembled plates immediately up to a height that is 2 mm below the position marked in step 2.3.
7. Gently layer 2-propanol onto the gel solution to flatten the top of separating gels.
8. Let the gels stand for 30 min at room temperature. It is not necessary to protect the gels from light.
   NOTE: It might take longer for gels to set under cold room temperature. Degassing the gel mixture before adding TEMED and APS helps accelerate this step. For this purpose, the gel mixture can be prepared in a 100-200 mL Erlenmeyer flask connected to a suction. Degas the solution for 10 min.
9. Remove the layered 2-propanol by absorbing with a paper towel.
10. Wash the top space of the gels by filling the space up with distilled water from a wash bottle and discard the water by pouring off into basin. Repeat washing 3 times.
11. Remove residual water remaining in the top space of the gels by absorbing with a paper towel.
12. Prepare 3 mL of 4% acrylamide gel mixture (4% (w/v) acrylamide (a: crylamide:bis-acrylamide = 29:1), 125 mM Tris-HCl (pH 6.8), 0.1% (w/v) SDS) in a 15 mL tube.
    NOTE: Do not add P-tag acrylamide or MnCl₂ solution to the stacking gel mixture.
13. Add 7.5 µL of TEMED and 24 µL of 10% (w/v) APS at final concentrations of 0.25% (v/v) and 0.08% (w/v), respectively. Mix well by gently swirling the tube for 5 s and pour the mixture on top of the separation gel. Immediately put appropriate combs (17-well plastic combs which accommodate up to 25 µL of samples, for example).
14. Let the gels stand for 30 min at room temperature. It is not necessary to protect the gels from light. After the stacking gels set, run them without further storage.

3. SDS–PAGE and Immunoblotting

1. Remove the combs from the gels. Then remove the silicon spacers and then clips from the gels.
2. Put the casted gels into gel tanks and fix the gel to the tank by clamping with the binder clips.
3. Pour the running buffer (25 mM Tris, 192 mM Glycine, 0.1% (w/v) SDS) at the bottom and top of the gels. Clean wells by flushing the running buffer using a 5 mL syringe and a 21G needle to remove gel pieces.
4. Remove air bubbles from the bottom space of the gels using a bent needle attached to a syringe. To make a bent needle, manually bend a 21G needle in the middle of the needle so that the angle between the tip and the base of the needle becomes 30-45 °.
5. Spin down precipitates caused by the addition of MnCl₂ at 20,000 × g for 1 min at room temperature to obtain clear samples.
6. Load 10 µg proteins for detecting the phosphorylation of overexpressed Rab10 and 30 µg proteins for endogenous Rab10.
   NOTE: It is critical to load equal volume of samples in ALL wells. Empty lanes should be loaded with 1× Laemmli's SDS-PAGE sample buffer. If the samples contain MnCl₂, add the same concentration of MnCl₂ to the dummy samples.
7. The well loaded with a molecular weight marker (MWM) should also be supplemented with 1× Laemmli's SDS-PAGE sample buffer so the volume of samples loaded is the same in all wells.
   NOTE: Again, if the samples contain MnCl₂, add the same concentration of MnCl₂ to the MWM. Alternatively, an EDTA-free MWM can be used.
8. Run gels at 50 V for stacking (approximately 30 min) until the dye front crosses into the separation gel.
9. After the samples stack, change the voltage to 120 V for separation until the dye front reaches the bottom of the gels (approximately 50 and 80 min for 80 and 100 mm long gels, respectively).
   NOTE: The migration pattern of the MWM is expected to be different from that on normal SDS-PAGE gels. It should not be used for estimating the molecular weight of proteins on P-tag SDS-PAGE gels but can be used for checking the reproducibility of P-tag gels. Refer to Discussion for details.
10. Wash the gels to remove MnCl₂ from the gels.
    1. Pour the transfer buffer (48 mM Tris, 39 mM Glycine, 20% (v/v) methanol) containing 10 mM EDTA and 0.05% (w/v) SDS into a container (e.g., large weighing boats). The volume of the buffer should be sufficient to cover a gel.
    2. Remove the separation gels from the gel plates and put one gel in one container. Discard the stacking gel.
    3. Leave the gels on a rocking shaker (~60 rpm) for 10 min at room temperature.
    4. Repeat the wash steps in total 3 times.
       NOTE: Use fresh buffer for each wash.
    5. Wash the gels once for 10 min with the transfer buffer containing 0.05% (w/v) SDS to remove EDTA. The volume of the buffer should be sufficient to cover a gel.
11. Electro-transfer to nitrocellulose or polyvinylidene difluoride (PVDF) membranes using wet tanks.
    1. Place a filter paper (10 x 7 cm) on a sponge pad for transfer. Place the gel on the filter paper. Make sure there are no air bubbles between the filter paper and the gel.
    2. Put a membrane (10 x 7 cm) on the gel and make sure there are no air bubbles between the gel and the membrane.
    3. Put another filter paper (10 x 7 cm) on the membrane and, again, make sure there are no air bubbles between the membrane and the filter paper.
    4. Put another sponge pad on the filter paper. Put the stack of membrane/filter papers in a cassette for transfer.
    5. Put the cassette into a transfer tank, making sure that the membrane is located between the gel and the positively charged anode.
    6. Connect the tank to a power supply and put the tank in a styrene foam box filled with ice-cold water. Start transfer at 100 V for 180 min.
       NOTE: Prolonged transfer is necessary since the transfer of proteins from P-tag SDS-PAGE gels are not as efficient as that from normal SDS-PAGE gels. Efficient cooling is essential to avoid melting gels during transfer.
12. Check the transfer
    1. Remove the membranes from the gels using tweezers and soak the membranes in a Ponceau S solution (0.1% (w/v) Ponceau S, 5% (v/v) acetic acid) to stain transferred proteins on the membranes in a plastic container. The volume of the solution should be sufficient to cover a membrane.
    2. Incubate the membranes for 1 min at room temperature by rocking manually.
       NOTE: A ladder of bands should become visible in each lane.
    3. Pick the membrane up out of the staining solution with tweezers and see if the ladder of bands has uniform pattern and intensity in every lane where the samples have been loaded.
    4. After checking the staining, remove the staining solution and add TBST buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% (v/v) polyoxyethylenesorbitan monolaurate). The volume of the buffer should be sufficient to cover a membrane.
       NOTE: The Ponceau S solution can be collected and re-used several times.
    5. Rock the membranes in TBST on a rocking shaker (~60 rpm) at room temperature until no visible bands remain on the membranes.
    6. Repeat the washing step with fresh TBST for 5 min.
13. Remove and discard the TBST. Block by adding 5% (w/v) skim milk dissolved in TBST and rocking on a shaker (~60 rpm) for 1 h at room temperature. The volume of the blocking solution should be sufficient to cover a membrane.
14. Prepare primary antibody solutions by diluting primary antibodies (anti-Rab10 antibody for endogenous Rab10 and anti-HA antibody for overexpressed HA-Rab10) in 10 mL per membrane of the blocking solution (see Table of Materials for concentrations).
15. Remove and discard the blocking solution and add the primary antibody. Incubate the membranes on a rocking shaker (~60 rpm) overnight at 4 °C.
16. Remove the primary antibody solution and add TBST to wash the membranes. The volume of the buffer should be sufficient to cover a membrane.
17. Incubate the membranes for 5 min on a rocking shaker (~60 rpm) at room temperature. Repeat the wash (step 3.16) in total 3 times using fresh TBST each time.
18. Prepare secondary antibody solutions by diluting secondary antibody labeled with horseradish peroxidase (HRP) in 10 mL per membrane of the blocking solution. Use anti-rabbit IgG antibody labeled with HRP for membranes probed with the anti-Rab10 antibody, and anti-mouse IgG antibody labeled with HRP for those probed with the anti-HA antibody (see Table of Materials for concentration).
19. Remove and discard the TBST after the third wash and add the secondary antibody solution. Incubate the membranes on a rocking shaker (~60 rpm) for 1 h at room temperature.
20. Wash the membranes in TBST for 10 min. Repeat the wash step in total 3 times, similar to step 3.17.
21. Develop the membranes using enhanced chemiluminescence (ECL).
    NOTE: Exposure time might vary depending on the ECL solution and the system used for detection of chemiluminescence.
    1. Turn on an imager equipped with a charge-coupled device (CCD) camera and a computer connected to the imager. Start up the control software for the imager. Wait until the temperature of the CCD camera has reached -25 °C.
    2. Put 1 mL of the ECL solution for one membrane on a plastic wrap spread on a bench.
    3. Put a membrane gel-side-up on the ECL solution and then quickly flip it over so that both sides of the membrane are coated with the solution.
    4. Pick the membrane up and drain it by letting one side of the membrane touch a paper towel for 5 s.
    5. Put the membrane between clear films (e.g., clear paper pockets).
       NOTE: Plastic wrap is not recommended for wrapping the membranes. The clear films used for wrapping the membrane need to be as flat as possible without visible wrinkles to avoid uneven background.
    6. Place the membrane on a black tray. Put the tray in the imager and close the door.
    7. Click the "Focusing" button in the window of the control software. Check that the membrane is correctly positioned. Click the "Return" button.
    8. Select "Precision" for "Exposure Type". Select "Manual" for "Exposure Time" and set the exposure time to 1 s.
    9. Select "High" for "Sensitivity/Resolution". Leave "Add Digitization Image" unchecked. Click the "Start" button to take an image.
    10. Save the image that appeared on the display in the computer as a TIFF file.
    11. Repeat taking images with the exposure time from 1, 3, 10, 30, 60, 90, 120, 150 s and up to 180 s. When taking the last image, check "Add Digitization Image" so the digital image, not the chemiluminescence, of the membrane can be simultaneously taken.
    12. Select the best image with the largest dynamic range and without any saturating pixels (shown in red) in the bands of interest.
        NOTE: Conventional X-ray films can also be used for detection¹⁹.

结果

Overexpression System: Phosphorylation of HA-Rab10 by 3×FLAG-LRRK2 in HEK293 Cells:

HEK293 cells were transfected with 0.266 µg of HA-Rab10 wild-type and 1.066 µg of 3×FLAG-LRRK2 (wild-type, kinase-inactive mutant (K1906M), or FPD mutants). Rab10 phosphorylation was examined by P-tag SDS-PAGE followed by immunoblotting using an anti-HA antibody (Figure 2). 10 µg of proteins were run on a 10% gel (80 x ...

讨论

Here, we describe a facile and robust method of detecting Rab10 phosphorylation by LRRK2 at endogenous levels based on the P-tag methodology. Because the currently available antibody against phosphorylated Rab10 works only with overexpressed proteins¹⁵, the present method utilizing P-tag SDS-PAGE is the only way to assess endogenous levels of Rab10 phosphorylation. Moreover, the present method allows the estimation of the stoichiometry of Rab10 phosphorylation in cells. Because the P-tag methodolo...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
<强>试剂
Dulbecco磷酸盐缓冲盐水（DPBS）	自制		150 mM NaCl、8 mM Na2HPO4-12H2O、2.7 mM KCl、1.5 mM KH2PO4 MilliQ水中，并通过高压灭菌
钠	Nacalai灭菌Tesque	31320-34
磷酸氢二钠钠 12-水	和光	196-02835
氯化钾	和光	163-03545
磷酸二氢钾	和光	169-04245
2.5% 胰蛋白酶 （10X）	Sigma-Aldrich	T4549	用无菌 DPBS 稀释 10 倍，用于制备工作溶液
Dulbecco 改良的 Eagle 培养基 （DMEM）	Wako	044-29765
胎牛血清	BioWest	S1560	在 56 °C 下热灭活;C 30 分钟
青霉素-链霉素 （100X）	Wako	168-23191
HEPES	Wako	342-01375
氢氧化钠	Wako	198-13765
聚乙亚胺盐酸盐 MAX，线性，分子量 40,000 （PEI MAX 40000）	PolySciences， Inc.	在 20 mM HEPES-NaOH （pH 7.0） 中制备 1 mg/mL 的储备液	24765-1
二甲基亚砜	Wako	045-28335
Tris	STAR	RSP-THA500G
盐酸	Wako	080-01066
聚氧乙烯 （10） 辛基苯醚	Wako	160-24751	相当于 Triton X-100
乙二醇-双（2-氨基乙基醚）-N，N，N'，N'-四乙酸 （EGTA）	和光	346-01312
原钒酸钠（V）	和光	198-09752
氟化钠	关东化学	37174-20
&β;-甘油磷酸二钠盐 五水合	Nacalai Tesque	17103-82
钠焦磷酸盐十水合	国产化学	2113899
微囊藻毒素-LR	Wako	136-12241
蔗糖	Wako	196-00015
完全不含 EDTA 的蛋白酶抑制剂混合物	罗氏	11873580001	一片溶解在 1 mL 水中，可在 -20 °C 下储存;C 的一个月。以 1：50 稀释度用于细胞裂解
Pierce 考马斯 （Bradford） 蛋白质检测试剂盒	Thermo Fisher Scientific	23200
十二烷基硫酸钠	Nacalai Tesque	31607-65
甘油	Wako	075-00616
溴酚蓝	Wako	021-02911
&β;-巯基乙醇	关东化学	25099-00
乙醇	Wako	056-06967
甲醇	Wako	136-01837
磷酸盐结合标签丙烯酰胺	Wako	AAL-107	P 标签丙烯酰胺
40% （w/v） 丙烯酰胺溶液	Nacalai Tesque	06119-45	丙烯酰胺：Bis = 29：1
四甲基乙二胺 （TEMED）	Nacalai Tesque	33401-72
过硫酸铵粉末溶解在 MilliQ 水中来制备	硫酸铵 （APS）	Wako	016-08021
2-丙醇	和光	166-04831
氯化锰四水合物	Sigma-Aldrich	M3634
Precision Plus 蛋白质预染标准	Bio-Rad	1610374、1610373 1610377	方案中使用的分子量标记
WIDE-VIEW 预染蛋白大小标记物 III	Wako	230-02461
甘氨酸	Nacalai Tesque	17109-64
Amersham Protran NC 0.45	GE Healthcare	10600007	硝酸纤维素膜
Durapore 膜过滤器	EMD Millipore	GVHP00010	PVDF 膜
滤纸 No.1	Advantec	00013600
Ponceau S	Nacalai Tesque	28322-72
乙酸	Wako	017-00251
Tween-20	Sigma-Aldrich	P1379	聚氧乙烯脱水水梨醇单月桂酸
乙二胺四乙酸酯 （EDTA）	Wako	345-01865
脱脂奶粉	Difco Laboratories	232100
Immunostar	Wako	291-55203	ECL 溶液（正常灵敏度）
Immunostar LD	Wako	290-69904	ECL 溶液（高灵敏度）
CBB 染色溶液自制			1 g CBB R-250、50% （v/v） 甲醇、10% （v/v） 乙酸，溶于 1 L MilliQ 水中
CBB R-250	Wako	031-17922
CBB 脱色			的 12% （v/v） 甲醇、7% （v/v） 乙酸的 1 L MilliQ 水
溶液名称	Company	目录号	评论
抗体
抗 HA 抗体	Sigma-Aldrich	11583816001	在 0.2 &mu 使用;g/mL，用于免疫印迹。
抗 Rab10 抗体	Cell Signaling Technology	#8127	以 1：1000 的比例用于免疫印迹. 特异性已在 Ito 等人中通过 CRISPR KO 确认，Biochem J，2016 年。
抗 pSer935 抗体	Abcam	ab133450	用于 1 μg/mL，用于免疫印迹。
抗 LRRK2 抗体	Abcam	ab133518	用于 1 μg/mL，用于免疫印迹。
抗 α;-微管蛋白抗体	Sigma-Aldrich	T9026	在 1 &mu 使用;g/mL，用于免疫印迹。
抗 GAPDH 抗体	Santa-Cruz	sc-32233	用于 0.02 μg/mL，用于免疫印迹。
过氧化物酶 AffiniPure 绵羊抗小鼠 IgG （H+L）	Jackson ImmunoResearch	515-035-003	在 0.16 &μ 使用;g/mL，用于免疫印迹。
过氧化物酶 AffiniPure 山羊抗兔 IgG （H+L）	Jackson ImmunoResearch	111-035-003	用于 0.16 &μ;g/mL，用于免疫印迹。
名称	>公司	目录号	注释
抑制剂
GSK2578215A	MedChem Express	HY-13237	储备液在 DMSO 中制备 10 mM 并储存在 -80 °C;C
MLi-2	由 Dario Alessi 博士（邓迪大学）提供储备		液在 DMSO 中制备 10 mM 并储存在 -80 °C;C
名称	>公司<	strong>目录号	评论
质粒
Rab10/pcDNA5 FRT 到 HA	由 Dario Alessi 博士提供 （邓迪大学）		该质粒表达氨基末端 HA 标记的人 Rab10。
LRRK2 WT/p3xFLAG-CMV-10	由 Takeshi Iwatsubo 博士（东京大学）提供Ito		等人，生物化学，46：1380–1388 (2007).该质粒表达氨基末端 3xFLAG 标记的野生型人 LRRK2。
LRRK2 K1906M/p3xFLAG-CMV-10	由 Takeshi Iwatsubo 博士（东京大学）提供，		生物化学，46：1380–1388 (2007).该质粒表达人 LRRK2 的氨基末端 3xFLAG 标记的 K1906M 激酶失活突变体。
LRRK2 N1437H/p3xFLAG-CMV-10			本文。该质粒表达人 LRRK2 的氨基末端 3xFLAG 标记的 N1437H FPD 突变体。
LRRK2 R1441C/p3xFLAG-CMV-10	由 Takeshi Iwatsubo 博士（东京大学）		Kamikawaji 等人提供，生物化学，48：10963–10975 (2013).该质粒表达人 LRRK2 的氨基末端 3xFLAG 标记的 R1441C FPD 突变体。
LRRK2 R1441G/p3xFLAG-CMV-10	由 Takeshi Iwatsubo 博士（东京大学）		Kamikawaji 等人提供，生物化学，48：10963–10975 (2013).该质粒表达人 LRRK2 的氨基末端 3xFLAG 标记的 R1441G FPD 突变体。
LRRK2 R1441H/p3xFLAG-CMV-10	由 Takeshi Iwatsubo 博士（东京大学）		Kamikawaji 等人提供，生物化学，48：10963–10975 (2013).该质粒表达人 LRRK2 的氨基末端 3xFLAG 标记的 R1441H FPD 突变体。
LRRK2 R1441S/p3xFLAG-CMV-10			本文。该质粒表达人 LRRK2 的氨基末端 3xFLAG 标记的 R1441S FPD 突变体。
LRRK2 Y1699C/p3xFLAG-CMV-10	由 Takeshi Iwatsubo 博士（东京大学）		Kamikawaji 等人提供，生物化学，48：10963–10975 (2013).该质粒表达人 LRRK2 的氨基末端 3xFLAG 标记的 Y1699C FPD 突变体。
LRRK2 G2019S/p3xFLAG-CMV-10	由 Takeshi Iwatsubo 博士（东京大学）		Kamikawaji 等人提供，生物化学，48：10963–10975 (2013).该质粒表达人 LRRK2 的氨基末端 3xFLAG 标记的 G2019S FPD 突变体。
LRRK2 I2020T/p3xFLAG-CMV-10	由 Takeshi Iwatsubo 博士（东京大学）		Kamikawaji 等人提供，生物化学，48：10963–10975 (2013).该质粒表达人 LRRK2 的氨基末端 3xFLAG 标记的 I2020T FPD 突变体。
名称	公司	目录号	评论
设备
CO2 培养箱	Thermo Fisher Scientific	Forma Series II 3110水套
式自动移液器	Drummond	Pipet-Aid PA-400
微量移液器 P10	Nichiryo	00-NPX2-10	0.5–10 μL
微量移液器 P200	Nichiryo	00-NPX2-200	20–200 亩;L
微量移液器 P1000	Nichiryo	00-NPX2-1000	100–1000 μL
微量移液器吸头 P10	STAR	RST-481LCRST
微量移液器无菌吸头 P200	FUKAEKASEI	1201-705YS
微量移液器无菌吸头 P1000	STAR	RST-4810BRST	无菌
5 mL 可分散移液器	Greiner	606180	无菌
10 mL 可分散移液器	Greiner	607180	无菌
25 mL 可散孢移液器	Falcon	357535	无菌
血细胞计数器	Sunlead Glass	A126	改进的 Neubeuer
显微镜	Olympus	CKX53
10 cm 培养皿	Falcon	353003	用于组织培养
6 孔板	AGC Techno Glass	3810-006	用于组织培养
涡旋混合器	Scientific Industries	Vortex-Genie 2
细胞刮刀	住友电木	MS-93100
1.5 mL 管	STAR	RSV-MTT1.5
15 mL 管	AGC Techno Glass	2323-015
50 mL 管	AGC Techno Glass	2343-050
离心机	TOMY	MX-307
96 孔板	Greiner	655061	不适用于组织培养
读板器	Molecular Devices	SpectraMax M2e
SDS–PAGE 储液槽	Nihon Eido	NA-1010
转运储液槽	Nihon Eido	NA-1510B
凝胶板（缺口）	Nihon Eido	NA-1000-1
凝胶板（普通）	Nihon Eido	NA-1000-2
硅垫片	Nihon Eido	NA-1000-16
17 孔梳	Nihon Eido	定制
活页夹	Nihon Eido	NA-1000-15
5 mL 注射器	Terumo	SS-05SZ
21G	Terumo	NN-2138R
发电站 1000 VC	ATTO	AE-8450	SDS 电源–页面和转移
大型称重船	Ina Optika	AS-DL
塑料容器	AS ONE	PS CASE No.4	10 x 80 x 50 毫米
摇床	Titech	NR-10
苯乙烯泡沫箱	通用		内部尺寸应适合一个转运罐（200 x 250 x 250 毫米）。
ImageQuant LAS-4000	GE Healthcare		配备制冷型 CCD 相机的成像仪，用于检测 ECL