Chemiluminescence and fluorescence imaging system

SW982 cells were cultured with different concentrations of BAI for 48 hours. Cell was lysed with RIPA buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM Na₃VO₄, and a protease inhibitor cocktail; Sigma-Aldrich). Cell lysates were measured Bradford protein assay kit (Bio-Rad) and were loaded on 8% or 10% polyacrylamide gels and electrophoresed in SDS-PAGE. Proteins were transferred to a PVDF membrane (Millipore, Eschborn, Germany) and then was blocked with 5% skim milk and immunoblotted with a primary antibody (Cell Signaling Technology, USA). Proteins were detected by enzyme-linked chemiluminescence (ECL) and quantified with a chemiluminescence and fluorescence imaging system (Bio-Rad, USA).

Total protein was extracted by using RIPA lysis buffer (NCM Biotech) with protease inhibitor cocktail and phosphatase inhibitor after transfection for 72 hours. 16-30 μg protein was loaded and electrophoresed on 8-12% SDS-PAGE gels, then transferred to a PVDF membranes (Millipore, Bedford, MA, USA). After sealing in NcmBlot blocking buffer (NCM Biotech) for 15 min, the membrane was incubated within primary antibody overnight at 4 °C. Then a horseradish peroxidase-conjugated secondary antibody (1:2000, Cell Signaling Technology) was used. Target protein bands were visualized using super sensitive ECL luminescence reagent (Dalian Meilun Biotechnology Co, Ltd.) in a Chemiluminescence and Fluorescence Imaging System (Bio-Rad). The antibodies used in this study were listed in Table S2.