SDS-PAGE and Immunoblot Analysis
SDS-PAGE/Western blot analysis was carried out as previously described
(J. Xie et al., 2019). Briefly, cells were lysed in ice-cold lysis
buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl pH 7.5,
2.5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium
orthovanadate, 1 mM dithiothreitol (DTT) and 1 mM β-glycerophosphate
supplemented with protease inhibitor cocktail. Protein concentrations
were determined via Bradford assay and normalized. Equal aliquots of
protein (20 µg) were denatured in Laemmli loading buffer, heated at 95°C
for 3 min and separated by SDS-PAGE using gels containing 7-13%
acrylamide and 0.1%-0.36% bis-acrylamide as required. Proteins were
transferred to nitrocellulose membranes, which were blocked and
incubated with primary antibody as indicated (Supplementary Table S1).
The relevant fluorescently-conjugated secondary antibody was applied and
signals were imaged using a LiCor Odyssey® CLx imager (Millennium
Science, VIC, Australia).