Ab167418

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.