Class 4 β tubulin

Total protein lysates were isolated from growing cells using 1x radioimmunoprecipitation assay buffer [RIPA, 1% (v/v) NP40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS in 1x PBS buffer] with freshly added protease inhibitors (cocktail from Bio-Rad Laboratories, Hercules, CA). Total protein (10–25 μg) was separated by 4–20% (w/v) gradient polyacrylamide gels and transferred onto nitrocellulose membranes using the Trans-Blot Turbo transfer system (all Bio-Rad Laboratories). Membranes were blocked overnight at 4 °C in 1x TBS containing 5% (w/v) nonfat milk and 1% (w/v) bovine serum albumin, and then incubated with the following antibodies: anti-P-gp, BCRP, and MRP2 (Signet Laboratories, Dedham, MA), anti-MRP7 (Thermo Fisher Scientific, Rockford, IL), anti-class I, pan α- and β-tubulin (Sigma Aldrich), anti-class II and III β-tubulin (Covance, Berkeley, CA), class IV β-tubulin (Abcam, Cambridge, MA), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), and specific antibodies for BRCA1, Bcl2, inhibitors of apoptosis, and p21 (Cell Signaling Technology, Danvers, MA). These primary antibodies were recognized by species-appropriate horseradish peroxidase-conjugated secondary antibodies, and detected using the Clarity Western ECL substrate (Bio-Rad).