Protease and phosphatase inhibitor cocktails

Total proteins of cells were extracted using RIPA lysis buffer (Beyotime, China) supplemented with protease and phosphatase inhibitor cocktails (TargetMol, United States) on ice, and the protein concentrations were then determined with a BCA assay (Pierce, United States). Twenty micrograms of protein was run on a 10–15% polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Germany). The blots were blocked with 5% BSA in TBS +0.1% tween 20 and incubated with primary antibodies (Supplementary Table S2) in 5% BSA overnight at 4°C. Then, the cells were incubated with the corresponding HRP-conjugated secondary antibodies (Proteintech, United States) for 1 h at room temperature. Images of the blots were obtained by using a Tanon system with enhanced chemiluminescence (ECL) reagent (Thermofisher, United States).

Cells were harvested and homogenized in RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitor cocktails (TargetMol, USA) on ice and subjected to a previously described protocol [3 (link)]. The signal intensities of bands obtained were analyzed by ImageJ (NIH, USA). The antibodies used are listed in Additional file 6: Table S2.