*4.8. Immunoprecipitation*

For exogenous immunoprecipitation, HEK293T cells were cotransfected with pcDNA3.0-Flag or Flag-KLF5 in the same vector with pSG5-AR plasmid using the Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer's protocol. After 48 h, the cells were collected and resuspended in cell lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 0.8% NP-40, 0.2% Triton X-100, and 3% glycerol. EDTA-free Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and PMSF (Sangon Biotech) were added to the cell lysis buffer. Cell lysates were incubated with anti-FLAG-agarose beads at 4 ◦C for 2 h (Sigma). Beads were washed and eluted and supernatants analyzed by western blotting. For endogenous immunoprecipitation, C4-2B cells or mouse prostate were collected and resuspended in lysis buffer and then rabbit or mouse normal IgG, KLF5 antibody (self-made) or AR antibody (06-680-AF488, Millipore) added to the lysate, followed by overnight incubation at 4 ◦C. Lysates coupled with antibody were incubated with magnetic Dynabeads (Invitrogen) for 2 h. Beads were washed extensively and eluted and supernatants analyzed by western blotting as above.
