Anti fkbp52

Co-immunoprecipitations were performed from extracts of LNCaP cells that were transfected with FKBP52 siRNA or 293T cells that were co-transfected with Gal4-AR LBD and β-catenin expression plasmids. Transfected or co-transfected cells maintained in 10% Charcoal-stripped serum were treated with and without 10 nM DHT and 30μM MJC13 following transfection, and harvested in RIPA cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.5 mM EGTA, 1% NP-40, Protease inhibitor cocktail (Roche)). Whole-cell lysate was incubated with 2 μg of anti-β-catenin or anti-Gal4 antibody (Santa Cruz Biotechnology) for 4 hours at 4ºC, and was further incubated for another 12 hours after the addition of 30 μl of protein A/G agarose bead slurry (Santa Cruz Biotechnology). Agarose beads were washed three times with RIPA buffer at 4ºC, and bound proteins were separated by SDS-PAGE. Proteins on the gels were transferred to a PVDF membrane, subjected to Western blot analysis with anti-AR (Santa Cruz Biotechnology), anti-FKBP52 (Cell Signaling Technologies), anti-β-catenin (Santa Cruz Biotechnology), and anti-GAPDH (Cell Signaling Technologies) antibodies, and then detected with an ECL kit (Amersham Pharmacia).

Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membrane using the Invitrogen Nu-Page system (Carlsbad, CA). Western blot analysis was performed using the following protein-/phospho-protein-specific antibodies: anti-p70 S6 kinase (#2708), anti-phospho-p70 S6 kinase (T389) (#9234), anti-rpS6 (#2217), anti-phospho-rpS6(S240/244) (#5364), anti-phospho-NDRG-1 (T346) (#5482), anti-NDRG-1 (#9485), anti-phospho-Akt (S473) (#4060), anti-Akt (#4691), anti-FKBP51 (#12210), anti-FKBP52 (#11826), anti-mTOR (#2972), anti-Rictor (#9476), anti-phospho-Rictor (T1135) (#3806), and anti-Raptor (#2280) were from Cell Signaling Technologies (Danvers, MA). Anti-FKBP25 (ab16654) and anti-FKBP12 (ab2918) were from Abcam. Anti-GAPDH (AM4300) was from Ambion Austin, TX.