Ecl western blotting system

Western blot analysis was conducted as previously described (14 (link)). Briefly, protein extracts from harvested experimental cells were prepared using lysis buffer from Beyotime Biotechnology (Shanghai, China). Thirty micrograms of protein extracts were loaded onto a 12% SDS-PAGE gel, electrophoretically separated, and transferred to polyvinylidene membranes (Beyotime Biotechnology, Shanghai, China). After incubating the membrane with 5% non-fat milk blocking buffer at room temperature for 1 h, the membranes were incubated with primary antibody overnight at 4°C. After incubation with a horseradish peroxidase-conjugated secondary antibody (1:2,500, Promega, Madison, WI, USA) at room temperature for 1 h, the signal was obtained by using an ECL Western blotting system (Promega, Madison, WI, USA) and visualized and quantified using the Bio-Rad ChemiDoc MP system. The primary antibodies against CORO2A (Sigma, catalog # HPA041302, 1:1,000) were obtained from Sigma (Sigma Chemical Co., St. Louis, MO, USA). β-actin, also obtained from Sigma, was used as the loading control.

Protein extraction and Western blot analysis were performed as previously described with minor modifications [33 (link)]. Briefly, total cellular proteins were extracted from the harvested cells using a lysis buffer [62.5 mM Tris-HCl pH 6.8, 100 mM dithiothreitol (DTT), 2% SDS and 10% glycerol]. The protein concentrations were determined using the Bicinchonininc acid method with the Bio-Rad protein assay following the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). Cellular proteins were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Blots were incubated in blocking buffer (5% non-fat dry milk in Tris-buffered saline with 0.5% Tween, TBS-T) at room temperature for 2 h. After washing with TBS-T, the nitrocellulose was incubated with a specific antibody against MDR1 (ab170904, Abcam, CA, USA), β-actin (AC-15, Sigma Chemical Co., St. Louis, MO, USA), MRP4 (#12705, Cell Signaling, CA, USA), XPD (ab167418, Abcam, CA, USA), TOPIIA (ab52934, Abcam, CA, USA), or CUL4A (ab92554, Abcam, CA, USA) overnight at 4°C. Following the incubation with a horseradish peroxidase-conjugated secondary antibody, the signal was detected using an ECL Western Blotting System (Promega, Madison, WI, USA) and visualized and quantitated using the Bio-Rad ChemiDoc MP system.