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).