The membrane fractions of ATP13A2-transfected HEK293 cells and SHSY5Y cells were solubilized in lysis buffer supplemented with 1.5% DDM
and 0.3% CHS. Insoluble material was removed by ultracentrifugation
at 366,000 × g, 30 min at 4 °C. The samples were then injected into a
Superose 6 10/300 GL column (GE Life Sciences) connected to the
AKTAexplorer 10 XT FPLC system (GE Healthcare) at 4 °C, equilibrated
in a running buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 0.03%
DDM, and 0.0015% CHS. Fractions of 1 mL were collected, precipitated
with 10% trichloroacetic acid (TCA), and analyzed by Western blotting.
1.
Phosphorylation assay
Membrane fractions (50 μg) were added to the reaction buffer (2 mM
MgCl2, 5 mM NaN3, 17 mM HEPES, pH 6.5 or 7.4) with or without 20 mM
KCl. The reaction was initiated on ice by adding [γ-32P] ATP (2 μCi) and
stopped after 20 s with the stop solution (20% trichloroacetic acid and
10 mM phosphoric acid). After precipitation on ice for 20 min, samples
were centrifuged (20,000 × g,10 min, 4 °C). The pellet was washed with
ice-cold stop solution and distilled water and dissolved in sample
buffer comprising 2% SDS, 2.5% dithiothreitol, 10% glycerol, and
50 mM Tris-HCl, pH 6.8, and subjected to the 5% SDS-polyacrylamide
gel under acidic conditions at pH 6.057. The radioactivity was visualized
and quantified by digital autoradiography of the dried gel using
Typhoon FLA 9500.
2.
3.
4.
5.
6.
7.
8.
9.
Detection of phosphorylated α-synuclein in SH-SY5Y cells
The undifferentiated SH-SY5Y cells transiently and stably expressing αsynuclein and the differentiated SH-SY5Y cells by retinoic acid and BDNF
were treated with SCH28080, vonoprazan, and bafilomycin A1 for 48 h.
The cells were fixed with ice-cold methanol for 5 min, washed with Trisbuffered saline (TBS), and permeabilized with permeabilization buffer
containing 0.3% Triton X-100 and 0.1% BSA in TBS for 15 min at room
temperature. Non-specific binding of the antibody was blocked with 2%
BSA in TBS, and the cells were incubated with an anti-phosphorylated αsynuclein antibody (pSyn#64) (1:100) in Can Get Signal immunostain
(Toyobo) for 15 h at 4 °C. The cells were washed with TBS and incubated
with the Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:100) for
1 h at room temperature. Immunofluorescence images were visualized
by using a Zeiss LSM 700 laser scanning confocal microscope. The area
(dimension) of the fluorescent signal due to phosphorylated α-synuclein
was measured by Zen3.3 software (Zeiss).
10.
11.
12.
13.
14.
15.
16.
Human tissue procurement
Human normal gastric mucosa was obtained from the dissected stomach of a Japanese gastric cancer patient (Female, 67 years) in
accordance with the recommendations of the Declaration of Helsinki
and with ethics committee approval of the University of Toyama (No.
R2017085). Informed consent was obtained from the patient at
Toyama University Hospital.
17.
18.
19.
Statistical analysis
Results are shown as mean ± SEM. Differences between groups were
analyzed by one-way analysis of variance, and correction for multiple
comparisons was made by using Tukey’s multiple comparison test. A
comparison between the two groups was made by using two-tailed
unpaired Student’s t test. Statistically significant differences were
assumed at P < 0.05.
Reporting summary
20.
21.
22.
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
23.
Data availability
Source data are available as a Source Data file. Source data are provided with this paper.
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Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research
(KAKENHI) from Japan Society for the Promotion of Science (JSPS) (to
H.S. (22H02801), Ta.F. (20K07258), S.N. (21H03365), and T.S.
(22K06827)), by JSPS Core-to-Core Program, B. Asia-Africa Science
Platforms, Tamura Science & Technology Foundation, and Platform for
drug discovery, informatics, and structural life science.
Author contributions
Ta.F. and H.S. designed all experiments and wrote the paper. Ta.F., S.N.,
P.W., S.Z., A.Y., T.S., Y.T., and T.O. performed the experiments. Ts.F. and
H.T. gave conceptual advice. All authors reviewed the results and
approved the final version of the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-023-37815-z.
Correspondence and requests for materials should be addressed to
Takuto Fujii or Hideki Sakai.
Peer review information Nature Communications thanks Curtis Okamoto and the other, anonymous, reviewer(s) for their contribution to the
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