P27kip1 c 19

Cells were cultured on glass coverslips until of a suitable density and were fixed in 4% paraformaldehyde. The cells were permeabilised in 0.3% Triton x-100 and non-specific staining was prevented by incubation in 10% goat serum in PBS. The primary antibody, p27^Kip1 (C-19, Santa Cruz) was applied for 1 hour at room temperature, at a concentration of 1:100. The cells were washed and Cy2-conjugated anti-rabbit antibody (Jackson) was applied for 1 hour at room temperature, at a concentration of 1:200. The cells were washed and the coverslips were mounted using ProLong Gold Antifade reagent (Life Technologies) with DAPI. Slides were viewed using an Olympus BX51 upright microscope using a 40x 0.75 UPlanFLN objective and captured using a Coolsnap EZ camera (Photometrics) through MetaVue Software (Molecular Devices). Filter sets for DAPI (31000v2) and FITC (41001) were used. Images were processed and analysed using ImageJ (http://rsb.info.nih.gov/ij). To quantify nuclear stains of p27^Kip1, 5 random fields were imaged and the colour channels were separated. A minimum total of 1000 nuclei were counted per cell line (based on DAPI staining).

Cells harvested at different time points were lysed and total protein extracts were used for western blotting using antibodies specific for AR (US Biological, Salem, MA), PSA (Santa Cruz Biotechnology, Dallas, TX), Cbl (C-15) (Santa Cruz Biotechnology, Dallas, TX), TRAF6 (Millipore, Temecula, CA), p27Kip1 (C-19) (Santa Cruz, Biotechnology), IRAK1 (F-4) (Santa Cruz, Biotechnology), ZFAND1 (A-14) (Santa Cruz Biotechnology), FGD4 (Epitomics, Burlingame, CA), ABHD3 (Biorbyt, Cambrige, UK), DOK4 (C-16) (Santa Cruz Biotechnology), EGFR (1005) (Santa Cruz Biotechnology), VEGFA (A-20) (Santa Cruz Biotechnology), α-tubulin (Cell Signaling, Danvers, MA), GAPDH (Sigma-Aldrich, St. Louis, MO). Total extracts (30–50 μg) were directly mixed with Lammeli sample buffer and separated on SDS-PAGE. Immunoblotting was performed using appropriate primary and horseradish peroxidase conjugated respective secondary antibodies. Positive signals were detected using a chemiluminiscence ECL kit (Pierce, Rockford, IL).