The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.
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.
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 reported3,4,5. 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 FPD6,7,8.
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 patients10,11,12, 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.
1. Sample Preparation for the P-tag SDS–PAGE
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.
3. SDS–PAGE and Immunoblotting
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 100 x 1 mm) containing 50 µM P-tag acrylamide and 100 µM MnCl2. The exposure time for the P-tag gel (top panel) was 10 s using a standard ECL solution. Co-overexpression of LRRK2 with Rab10 caused band shift on the P-tag gel (marked with an open circle in the top panel; compare lanes 2 and 4). In contrast, co-expression with a kinase-inactive mutant (K1906M) LRRK2 failed to change the mobility of Rab10 (compare lanes 4 and 5), indicating that the band shift is due to the LRRK2 kinase activity. The band shift was increased by all FPD mutations (compare lanes 4 and 6-13) in agreement with the previous report15.
Endogenous System: Phosphorylation of Rab10 by LRRK2 in Cultured Cells:
Mouse 3T3-Swiss albino embryonic fibroblast cells were treated with or without a LRRK2 inhibitor (GSK2578215A)20 at a final concentration of 1 µM for 1 h (Figure 3A). Human lung carcinoma A549 cells were treated with or without another LRRK2 inhibitor (MLi-2)21 at a final concentration of 10 nM for 1 h (Figure 3B). Endogenous Rab10 phosphorylation by endogenous LRRK2 was examined by P-tag SDS-PAGE followed by immunoblotting with an anti-Rab10 antibody. 30 µg of proteins were run on a 10% gel (100 x 100 x 1 mm for 3T3-Swiss albino cells and 80 mm for A549 cells) containing 50 µM P-tag acrylamide and 100 µM MnCl2. The exposure for the P-tag gel (top panel) in Figure 3A and Figure 3B were 3 min and 90 s, respectively. The band shift corresponding to phosphorylated endogenous Rab10 was observed on the P-tag gel (marked with an open circle in the top panel; compare lanes 1-2 and 3-4) which disappeared upon treatment of cells with GSK2578215A or MLi-2. The efficacy of the LRRK2 inhibitors was validated by immunoblotting using the anti-pSer935 LRRK2 antibody (the third panel from the bottom), which is a well-established readout of LRRK2 inhibition in cells22.
Figure 1. Schematic depiction of P-tag SDS–PAGE. When a mixture of phosphorylated (red circles) and non-phosphorylated (blue circles) proteins (e.g., Rab10) runs through a gel where P-tag acrylamide is co-polymerized, the mobility of only phosphorylated proteins retards due to the strong interaction of phosphorylated proteins with the P-tag molecules (broken arrows). Since Rab10 is phosphorylated by LRRK2 at a single residue Thr73, Rab10 runs as two bands: the top band represents phosphorylated Rab10 and the bottom band represents non-phosphorylated Rab10. When cells are treated with LRRK2 inhibitors, the top band should disappear. Please click here to view a larger version of this figure.
Figure 2. Representative result of the P-tag blot: overexpression system. The top panel shows the P-tag blot using an anti-HA antibody, where phosphorylated and non-phosphorylated Rab10 are marked with open (○) and closed (●) circles, respectively. The panels shown below the P-tag blot are immunoblots on normal SDS-PAGE gels using an anti-HA, anti-FLAG, anti-α-tubulin, and anti-GAPDH antibodies. The HA blot and FLAG blot are shown for ensuring the overexpression of HA-Rab10 and 3xFLAG-LRRK2, respectively. The α-tubulin and GAPDH blots are shown for ensuring the equal loading. Please click here to view a larger version of this figure.
Figure 3. Representative result of the P-tag blot: endogenous system. Two cell lines, namely (A) 3T3-Swiss albino cells and (B) A549 cells, were used. In both figures, the top panels show the P-tag blots using an anti-Rab10 antibody, where phosphorylated and non-phosphorylated endogenous Rab10 are marked with open (○) and closed (●) circles, respectively. The panels shown below the P-tag blots are immunoblots on normal SDS-PAGE gels using an anti-Rab10, anti-pSer935 LRRK2, anti-LRRK2, and anti-α-tubulin antibodies. The Rab10 and LRRK2 are shown for ensuring the similar expression of endogenous Rab10 and LRRK2 between the samples, respectively. The pSer935 LRRK2 blot are shown for ensuring that GSK2578215A (A) and MLi-2 (B) worked as expected. The α-tubulin blot are shown for ensuring the equal loading. The images of the P-tag blots superimposed with the molecular weight marker are shown in Figure S4. Please click here to view a larger version of this figure.
Figure S1. DNA sequences of the plasmids. The DNA sequences of the plasmid encoding HA-Rab10/pcDNA5 FRT TO and that of 3×FLAG-LRRK2/p3×FLAG-CMV-10. All plasmids used in this protocol are available from the authors upon request. Please click here to download this file.
Figure S2. An example of the optimization of P-tag SDS-PAGE. The same set of samples as that used in Figure 3A was used for optimizing the P-tag SDS-PAGE. Four different conditions were tested as shown in the figure. The bands corresponding to phosphorylated and non-phosphorylated Rab10 are highlighted with solid and dashed line rectangles, respectively. Based on this experiment, the condition with 10% acrylamide, 50 µM P-tag acrylamide and 100 µM MnCl2 gave the best separation of the two bands, both locating in the middle of the gel. Please click here to download this file.
Figure S3. Comparison of the migration pattern of a molecular weight marker. The molecular weight marker (MWM) was run on a regular SDS-PAGE gel (left panel) or on a P-tag SDS-PAGE gel (middle panel), and the gels were stained by Coomassie staining. The right panel shows an immunoblot of the samples used in Figure 2 (lane 4 and 5) on the same P-tag SDS-PAGE gel as the middle panel to show the positions of phosphorylated and non-phosphorylated Rab10. The arrowhead on the right-hand side of the immunoblot indicates the position of one of the MWM on the P-tag SDS-PAGE gel. Please click here to download this file.
Figure S4. The position of a molecular weight marker on P-tag SDS-PAGE gels. Digitized images of the membranes used for Figure 3A and Figure 3B are shown as (A) and(B), respectively. The immunoblots shown in Figure 3 are superimposed to show the relative position of the molecular weight marker (MWM). The MWM did not transfer well to the membranes but the marker (arrowhead) in Figure S3 was faintly but consistently observed. The position of the MWM is marked with dotted lines. Note that lanes irrelevant to the figures between the MWM and the lanes of interest are crossed out. Please click here to download this file.
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 methodology is generically applicable to phospho-proteins, the present protocol can be a "prototype" for establishing similar methods for other phospho-proteins.
Critical steps in the protocol are casting gels and preparation of samples. P-tag acrylamide is a relatively photo-labile reagent and the ability to retard the electrophoretic mobility of phosphorylated proteins is sometimes lost after long term storage at 4 °C. Researchers should take every care to avoid exposing P-tag acrylamide to light. Moreover, the P-tag molecule needs to form a complex with Mn2+ ions to capture phosphates. Thus, samples should not contain chelating agents such as EDTA, which are usual components of commercially available SDS-PAGE sample buffers. We recommend using the classical Laemmli's sample buffer to prepare samples for P-tag SDS-PAGE.
Another critical point is to thoroughly optimize the concentrations of acrylamide, P-tag acrylamide and MnCl2 in the separation gel mixture (Figure S2). The migration distances of phosphorylated and non-phosphorylated bands will vary depending on the reagents used (lot, purity, etc.), and optimization of the proper concentrations by testing several different concentrations in combination is mandatory (e.g., P-tag acrylamide (25, 50, 75 µM), MnCl2 (1:2 or 1:3 molar ratio to P-tag acrylamide), and acrylamide (7.5, 10, 12.5%)). The phosphorylated and non-phosphorylated bands should appear in the middle of the gels and well separated from each other. For optimization process, it is recommended to use overexpressed Rab10 because the shifted band is readily detectable. The authors can provide control samples for this protocol.
One of the biggest confusion that this protocol can cause will be due to the difference of the migration patterns of the MWM between regular and P-tag SDS-PAGE gels. As shown in Figure S3, the migration patterns of the MWM on regular and P-tag SDS-PAGE gels (both 10%) are greatly different and one cannot use the MWM for estimating the molecular weight of proteins on P-tag SDS-PAGE gels. However, on P-tag SDS-PAGE gels, one of the MWM stands out by migrating to the middle of the gel (arrowhead in Figure S3), which is consistently observed among gels (see Figure S4). This MWM always migrates in between the bands of phosphorylated and non-phosphorylated Rab10. Therefore, this marker is useful not only for ensuring that P-tag SDS-PAGE works before transfer but also as a landmark for having a rough sense of where the bands of phosphorylated and non-phosphorylated Rab10 will be seen.
For detection of bands on immunoblots, we have used an imager equipped with a cooled CCD camera (see Table of Materials) as well as a conventional X-ray film developing system, and found that both systems work well. For detection of endogenous levels of Rab10 phosphorylation in cultured cells, it is necessary to use cell lines which have high expression levels of endogenous LRRK2, such as mouse embryonic fibroblasts (either primary cultured or immortalized cell lines (3T3-Swiss albino, etc.)) and human lung carcinoma-derived A549 cells. For detection of endogenous levels of Rab10 phosphorylation in mouse tissues, it is recommended to use lung16.
The quantitation of Rab10 phosphorylation is not beyond that of usual immunoblotting. If the stoichiometry of Rab10 phosphorylation is very low (as shown in Figure 3), the intensity of non-phosphorylated band (marked with a closed circle) tends to be far above the saturation level, making the precise determination of the stoichiometry impossible. Nevertheless, qualitative estimation is still possible in any case, which is not obtainable without using the present method. Since the detection of the phosphorylation of endogenous Rab10 requires prolonged exposure, there tends to be some nonspecific bands appearing on the P-tag blot even though the anti-Rab10 antibody used in this protocol gives fairly clean immunoblots for normal use. To distinguish phosphorylated proteins from nonspecific bands, it is critical to use an inhibitor-treatment control, in which phosphorylated bands, but not nonspecific bands, should disappear. LRRK2 phosphorylates Rab8 besides Rab1015, and we have successfully applied the present protocol to Rab8A as well (data not shown). The present protocol can potentially be used to examine the phosphorylation of any proteins, although there might be technical difficulties if the protein is large (>100 kDa) or multiply phosphorylated. Because Rab10 is a small protein (25 kDa) and singly phosphorylated at Thr73 by LRRK2, the result of P-tag SDS-PAGE is simple where only one shifted band is observed.
In the near future, it will be critical to establish the method to quantitatively detect the kinase activity of LRRK2 in patient-derived samples in a high-throughput manner to assess the alteration of the kinase activity of LRRK2 in PD patients as well as to evaluate the effect of drugs on LRRK2 in clinical trials. Since human peripheral blood mononuclear cells (PBMCs) express relatively high levels of endogenous LRRK223, PBMCs or further isolated blood cells will be worth testing for endogenous Rab10 phosphorylation. The present method will not only be useful in investigating the basic biology of the LRRK2 signaling pathway, but also aid in obtaining a basic proof-of-concept for deciding which sample in patient-derived samples is to be analyzed in such large-scale studies.
The authors have nothing to disclose.
We thank Dr. Takeshi Iwatsubo (University of Tokyo, Japan) for kindly providing the plasmids encoding 3xFLAG-LRRK2 WT and mutants. We also thank Dr. Dario Alessi (University of Dundee, UK) for kindly providing MLi-2 and the plasmid encoding HA-Rab10. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP17K08265 (G.I.).
Reagents | |||
Dulbecco's phosphate-buffered saline (DPBS) | homemade | 150 mM NaCl, 8 mM Na2HPO4-12H2O, 2.7 mM KCl, 1.5 mM KH2PO4 in MilliQ water and sterilized by autoclaving | |
Sodium chloride | Nacalai Tesque | 31320-34 | |
Sodium Disodium Hydrogenphosphate 12-Water | Wako | 196-02835 | |
Potassium chloride | Wako | 163-03545 | |
Potassium Dihydrogen Phosphate | Wako | 169-04245 | |
2.5% Trypsin (10X) | Sigma-Aldrich | T4549 | Dilute 10-fold with sterile DPBS for preparing working solution |
Dulbecco's modified Eagle medium (DMEM) |
Wako | 044-29765 | |
Fetal bovine serum | BioWest | S1560 | Heat-inactivated at 56 °C for 30 min |
Penicillin-Streptomycin (100X) | Wako | 168-23191 | |
HEPES | Wako | 342-01375 | |
Sodium hydroxide | Wako | 198-13765 | |
Polyethylenimine HCl MAX, Linear, Mw 40,000 (PEI MAX 40000) | PolySciences, Inc. | 24765-1 | Stock solution was prepared in 20 mM HEPES-NaOH pH 7.0 at 1 mg/mL and the pH was then adjusted to 7.0 with NaOH |
Dimethyl sulfoxide | Wako | 045-28335 | |
Tris | STAR | RSP-THA500G | |
Hydrochloric acid | Wako | 080-01066 | |
Polyoxyethylene(10) Octylphenyl Ether | Wako | 160-24751 | Equivalent to Triton X-100 |
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) | Wako | 346-01312 | |
Sodium orthovanadate(V) | Wako | 198-09752 | |
Sodium fluoride | Kanto Chemical | 37174-20 | |
β-Glycerophosphoric Acid Disodium Salt Pentahydrate | Nacalai Tesque | 17103-82 | |
Sodium pyrophosphate decahydrate | Kokusan Chemical | 2113899 | |
Microcystin-LR | Wako | 136-12241 | |
Sucrose | Wako | 196-00015 | |
Complete EDTA-free protease inhibitor cocktail | Roche | 11873580001 | Dissolve one tablet in 1 mL water, which can be stored at -20 °C for a month. Use it at 1:50 dilution for cell lysis |
Pierce Coomassie (Bradford) Protein Assay Kit | Thermo Fisher Scientific | 23200 | |
Sodium dodecyl sulfate | Nacalai Tesque | 31607-65 | |
Glycerol | Wako | 075-00616 | |
Bromophenol blue | Wako | 021-02911 | |
β-mercaptoethanol | Kanto Chemical | 25099-00 | |
Ethanol | Wako | 056-06967 | |
Methanol | Wako | 136-01837 | |
Phosphate-binding tag acrylamide | Wako | AAL-107 | P-tag acrylamide |
40% (w/v) acrylamide solution | Nacalai Tesque | 06119-45 | Acrylamide:Bis = 29:1 |
Tetramethylethylenediamine (TEMED) | Nacalai Tesque | 33401-72 | |
Ammonium persulfate (APS) | Wako | 016-08021 | 10% (w/v) solution was prepared by dissolving the powder of ammonium persulfate in MilliQ water |
2-propanol | Wako | 166-04831 | |
Manganese chloride tetrahydrate | Sigma-Aldrich | M3634 | |
Precision Plus Protein Prestained Standard | Bio-Rad | 1610374, 1610373, 1610377 | Molecular weight marker used in the protocol |
WIDE-VIEW Prestained Protein Size Marker III | Wako | 230-02461 | |
Glycine | Nacalai Tesque | 17109-64 | |
Amersham Protran NC 0.45 | GE Healthcare | 10600007 | Nitrocellulose membrane |
Durapore Membrane Filter | EMD Millipore | GVHP00010 | PVDF membrane |
Filter Papers No.1 | Advantec | 00013600 | |
Ponceau S | Nacalai Tesque | 28322-72 | |
Acetic acid | Wako | 017-00251 | |
Tween-20 | Sigma-Aldrich | P1379 | polyoxyethylenesorbitan monolaurate |
Ethylenediaminetetraacetic acid (EDTA) | Wako | 345-01865 | |
Skim milk powder | Difco Laboratories | 232100 | |
Immunostar | Wako | 291-55203 | ECL solution (Normal sensitivity) |
Immunostar LD | Wako | 290-69904 | ECL solution (High sensitivity) |
CBB staining solution | homemade | 1 g CBB R-250, 50% (v/v) methanol, 10% (v/v) acetic acid in 1 L of MilliQ water | |
CBB R-250 | Wako | 031-17922 | |
CBB destaining solution | homemade | 12% (v/v) methanol, 7% (v/v) acetic acid in 1 L MilliQ water | |
Name | Company | Catalog Number | Comments |
Antibodies | |||
anti-HA antibody | Sigma-Aldrich | 11583816001 | Used at 0.2 μg/mL for immunoblotting. |
anti-Rab10 antibody | Cell Signaling Technology | #8127 | Used at 1:1000 for immunoblotting. Specificity was confirmed by CRISPR KO in Ito et al., Biochem J, 2016. |
anti-pSer935 antibody | Abcam | ab133450 | Used at 1 μg/mL for immunoblotting. |
anti-LRRK2 antibody | Abcam | ab133518 | Used at 1 μg/mL for immunoblotting. |
anti-α-tubulin antibody | Sigma-Aldrich | T9026 | Used at 1 μg/mL for immunoblotting. |
anti-GAPDH antibody | Santa-Cruz | sc-32233 | Used at 0.02 μg/mL for immunoblotting. |
Peroxidase AffiniPure Sheep Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 515-035-003 | Used at 0.16 μg/mL for immunoblotting. |
Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 111-035-003 | Used at 0.16 μg/mL for immunoblotting. |
Name | Company | Catalog Number | Comments |
Inhibitors | |||
GSK2578215A | MedChem Express | HY-13237 | Stock solution was prepared in DMSO at 10 mM and stored at -80 °C |
MLi-2 | Provided by Dr Dario Alessi (University of Dundee) | Stock solution was prepared in DMSO at 10 mM and stored at -80 °C | |
Name | Company | Catalog Number | Comments |
Plasmids | |||
Rab10/pcDNA5 FRT TO HA | Provided by Dr Dario Alessi (University of Dundee) |
This plasmid expresses amino-terminally HA-tagged human Rab10. | |
LRRK2 WT/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Ito et al., Biochemistry, 46: 1380–1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged wild-type human LRRK2. | |
LRRK2 K1906M/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Ito et al., Biochemistry, 46: 1380–1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged K1906M kinase-inactive mutant of human LRRK2. | |
LRRK2 N1437H/p3xFLAG-CMV-10 | This paper. This plasmid expresses amino-terminally 3xFLAG-tagged N1437H FPD mutant of human LRRK2. | ||
LRRK2 R1441C/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441C FPD mutant of human LRRK2. | |
LRRK2 R1441G/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441G FPD mutant of human LRRK2. | |
LRRK2 R1441H/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441H FPD mutant of human LRRK2. | |
LRRK2 R1441S/p3xFLAG-CMV-10 | This paper. This plasmid expresses amino-terminally 3xFLAG-tagged R1441S FPD mutant of human LRRK2. | ||
LRRK2 Y1699C/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged Y1699C FPD mutant of human LRRK2. | |
LRRK2 G2019S/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged G2019S FPD mutant of human LRRK2. | |
LRRK2 I2020T/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged I2020T FPD mutant of human LRRK2. | |
Name | Company | Catalog Number | Comments |
Equipments | |||
CO2 incubator | Thermo Fisher Scientific | Forma Series II 3110 Water-Jacketed | |
Auto Pipette | Drummond | Pipet-Aid PA-400 | |
Micropipette P10 | Nichiryo | 00-NPX2-10 | 0.5–10 μL |
Micropipette P200 | Nichiryo | 00-NPX2-200 | 20–200 μL |
Micropipette P1000 | Nichiryo | 00-NPX2-1000 | 100–1000 μL |
Tips for micropipette P10 | STAR | RST-481LCRST | Sterile |
Tips for micropipette P200 | FUKAEKASEI | 1201-705YS | Sterile |
Tips for micropipette P1000 | STAR | RST-4810BRST | Sterile |
5 mL disporsable pipette | Greiner | 606180 | Sterile |
10 mL disporsable pipette | Greiner | 607180 | Sterile |
25 mL disporsable pipette | Falcon | 357535 | Sterile |
Hematocytometer | Sunlead Glass | A126 | Improved Neubeuer |
Microscope | Olympus | CKX53 | |
10 cm dishes | Falcon | 353003 | For tissue culture |
6-well plates | AGC Techno Glass | 3810-006 | For tissue culture |
Vortex mixer | Scientific Industries | Vortex-Genie 2 | |
Cell scrapers | Sumitomo Bakelite | MS-93100 | |
1.5 mL tubes | STAR | RSV-MTT1.5 | |
15 mL tubes | AGC Techno Glass | 2323-015 | |
50 mL tubes | AGC Techno Glass | 2343-050 | |
Centrifuges | TOMY | MX-307 | |
96-well plates | Greiner | 655061 | Not for tissue culture |
Plate reader | Molecular Devices | SpectraMax M2e | |
SDS–PAGE tanks | Nihon Eido | NA-1010 | |
Transfer tanks | Nihon Eido | NA-1510B | |
Gel plates (notched) | Nihon Eido | NA-1000-1 | |
Gel plates (plain) | Nihon Eido | NA-1000-2 | |
Silicon spacers | Nihon Eido | NA-1000-16 | |
17-well combs | Nihon Eido | Custom made | |
Binder clips | Nihon Eido | NA-1000-15 | |
5 mL syringe | Terumo | SS-05SZ | |
21G | Terumo | NN-2138R | |
Power Station 1000 VC | ATTO | AE-8450 | Power supply for SDS–PAGE and transfer |
Large weighing boats | Ina Optika | AS-DL | |
Plastic containers | AS ONE | PS CASE No.4 | 10 x 80 x 50 mm |
Rocking shaker | Titech | NR-10 | |
Styrene foam box | generic | The internal dimensions should fit one transfer tank (200 x 250 x 250 mm). | |
ImageQuant LAS-4000 | GE Healthcare | An imager equipped with a cooled CCD camera for detection of ECL |