2.5. Cell lysates and western blot studies

Cells were placed in a 4 °C RIPA lysis buffer containing a protease inhibitor mixture (Sigma). Thirty to fifty micrograms of protein were separated on 8%, 10%, or 12% SDS-PAGE gels, transferred to 0.45 μm nitrocellulose membranes (Thermo Scientific), and blocked with 5% nonfat dry milk in phosphate-buffered saline and 0.1% Tween 20 (PBS-T) or OneBlock Western-CL blocking buffer (Genesee Scientific; El Cajon, CA). Membranes were incubated with primary antibody overnight at 4C. The following antibodies were used: AR (N20) (Santa Cruz Biotechnology; Santa Cruz, CA) or A303 (Bethyl; Montgomery, TX), cleaved PARP (Cell Signaling; Danvers, MA), tubulin (Thermo Scientific), GAPDH (Santa Cruz Biotechnology). The following day, membranes were incubated with secondary antibody conjugated to HRP and development was carried out using SuperSignal West Femto chemoluminescence (Thermo Scientific) or were imaged using LI-COR near-infrared western blot detection. Loading was assessed either by GAPDH or tubulin.