Comprehensive Protein Analysis in PCa Cells

Western blot analysis was performed as previously described [3 (link)]. In brief, PCa cells were added in radio immunoprecipitation assay buffer (RIPA, KeyGEN, BioTECH, China), and BCA Protein Assay Kit (KeyGEN, BioTECH, China) was used for protein determination. Then, the proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in no fat milk and then incubated with primary antibodies overnight at 4 °C. The primary antibodies included anti-Tubulin antibody (Cell Signaling Technology, USA, 1:1000), anti-ARHGAP5 antibody (ABclonal, China, 1:1000), anti-HDAC4 antibody (ABclonal, China, 1:1000), anti-IGF2BP3 antibody (Abcam, USA, 1:1000), anti-ZEB-1 antibody (Cell Signaling Technology, USA, 1:1000), anti-E-Cadherin antibody (Cell Signaling Technology, USA, 1:1000), anti-Vimentin antibody (Cell Signaling Technology, USA, 1:1000), and phospho-Erk1/2-T202/Y204 antibody (ABclonal, China, 1:1000). Subsequently, the membranes were immersed with the secondary antibody for 1 h. Finally, enhanced chemiluminescence (ECL) kit (Pierce Biotechnology, USA) was used to detect the level of protein.