Ar v7

Whole-cell protein extracts were incubated for 16 h at 4^oC with the indicated primary antibodies AR (441), AR (N20), AR (C-19), HSP70 (F-3 and H-300), STUB1(H231 and G-2), HA (F-3), Ubiquitin (P4D1 and FL76), 1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA; STUB1 (C3B6, 1:100 for IP, Cell Signaling antibody); AR-V7 (AG10008, Mouse monoclonal antibody, 1:1000 dilution, precision antibody); FLAG® M2 monoclonal antibody (F1804, 1:1000 dilution for western blot, 1:200 for IP, Sigma-Aldrich, St. Louis, MO); Tubulin (T5168, Monoclonal Anti-α-Tubulin antibody, 1:5000 dilution, Sigma-Aldrich, St. Louis, MO). Following secondary antibody incubation, immunoreactive proteins were visualized with an enhanced chemiluminescence detection system (Millipore, Billerica, MA). The bands were quantified by ImageJ.

Cells were lysed with RIPA buffer supplemented with protease inhibitor tablets and phosphatase inhibitor cocktail. Protein concentration was quantified using Pierce BCA protein assay kit following manufacturer’s protocol. 40 micrograms of protein were loaded into GenScript SurePage 4%-12% polyacrylamide gel, transferred to both nitrocellulose and PVDF membranes, blocked in 5% BSA or 5% milk in 1x TBST for one hour, before incubating the membranes with primary antibodies overnight at 4°C in 1% BSA solution. Membranes were washed with 1x TBST 3 three times before incubating the membranes in LI-COR IR-conjugated secondary antibodies (1:10,000-20,000) for 2 hours at room temperature. Membranes were washed three times with 1x TBST and imaged using the LI-COR Odyssey System. Membranes were adjusted and quantified with the LI-COR Image Studio Lite software (v5.2). The following antibodies were used for Western Blot Analysis: At 1:1000 Total AR (rabbit CST: #5153), Total SRC (rabbit CST: #2109), Total β-actin (mouse Santa Cruz: 4970S), ARpY534 (rabbit Invitrogen: #PA5-64643), SRCpY416 (rabbit CST: #2101), FKBP5 (rabbit CST: #8245), NKX3.1 (rabbit CST: #83700) cleaved PARP (mouse CST: #32563), total PARP (rabbit CST: #9532). At 1:500, ARpS81 (rabbit Sigma-Aldrich: #07-1375), AR-V7 (rabbit CST: #19672). Blots are results in triplicate.