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. Author manuscript; available in PMC: 2014 Jun 1.
Published in final edited form as: Steroids. 2013 Feb 1;78(6):548–554. doi: 10.1016/j.steroids.2012.12.013

Figure 3. Stability of AR and V7 Protein.

Figure 3

(A–D) LNCaP-V7 cells were transferred to stripped serum and treated with vehicle (EtOH), 10 nM R1881, or 0.25 ng/mL Dox overnight followed by treatment of 10 μg/mL Cycloheximide and 1 μM GA for the indicated time points. Cells were harvested and AR, V7, and Tubulin protein expression was detected by western blot. In panels A and C, AR and V7 were analyzed from the same blot in each case, but slightly different exposures were used to optimize detection of changes in protein expression. Protein expression was quantified relative to each respective Tubulin. To calculate the ratios below each AR and V7 band, those values were normalized to the “0 hr” lane for each blot. GA accelerated AR protein degradation but not that of V7 (compare A versus C). Densitometry analysis using Image J software was performed to show average fold change across three independent experiments (B and D). (E) To examine the effect of Hsp90 inhibition on endogenous V7 protein, 22Rv1 cells were transferred to charcoal stripped serum and treated with vehicle (DMSO) or 1 μM GA for 24 hours. Cells were harvested, protein was isolated, and AR, V7, and Tubulin expression were analyzed by western blotting. GA decreased AR protein while endogenous V7 protein was unaffected.