Immunoprecipitation and Western Blot Analysis

For immunoprecipitation, the cells were washed by cold PBS twice before being lysed in IP lysis buffer supplemented with protease and phosphatase inhibitors. Total protein lysate (2 mg) was used for each immunoprecipitation using specific antibody, Protein A agarose beads (GE Healthcare Uppsala, Sweden) were added to the cells and incubated overnight 4°C. Washed precipitated proteins were analyzed by western blot. The immunoprecipitation (IP), western blot and GST pull-down assays used in this study have been described previously in detail [23 (link)]. The samples were incubated with anti-E-cadherin (BD), anti-PAK5 (R&D), anti-GATA1/-Myc (Santa Cruz), anti-pGATA1 S161 (Bioss), anti-GFP (Genscript), anti-fibronectin (Sigma), anti-Flag (Shang Hai Ruixing), anti-His (Genscript), anti-GAPDH (Kang Chen, as a loading control) antibodies.