Western Blot Analysis of Protein Expression

Western blot was performed as described previously (Wang, et al. 2012b (link)). Briefly, normal cell lines or treated KGN cells were harvested on ice with ice-cold cell lysis buffer containing 10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 1% triton X-100, 10% glycerol, 0.1% SDS and 0.5% deoxycholate and protease and phosphatase inhibitor cocktails. Protein (30 μg) was loaded to a 10% SDS-PAGE gel, fractioned with electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA and probed with appropriate primary and HRP-conjugated secondary antibodies. The immunosignal was detected with a Thermo Scientific SuperSignal West Femto Chemiluminescent Substrate Kit. The images were captured and analyzed with a UVP gel documentation system (UVP, Upland, CA).