P fak

Semi-confluent PC3 cells were rinsed twice with PBS and separated into single cells by treatment with trypsin/EDTA solution (HyClone Laboratories, Logan, UT, USA). Trypsin inhibitor from glycine max soybean (Sigma-Aldrich Co.) was added to deactivate the trypsin. The cells were pre-incubated with and without WEGST (50 µg/mL) for 30 min; 3 × 10⁵ cells were then seeded on collagen I (10 µg/mL)-coated plates and harvested at various times, as indicated. Total protein was extracted from each cell and transferred to PVDF membranes, as described above. The membranes were first used for the detection of phosphor-FAK expression using the phosphospecific antibody (p-FAK; BD Biosciences, San Jose, CA, USA), and the blot was stripped through incubation with 1 N NaOH during 1–2 min at room temperature; it was then washed three times with TBS-T and used for the detection of total FAK (t-FAK) using the mouse anti-FAK antibody (BD Biosciences, San Jose, CA, USA). The activation rate of FAK (p-FAK/t-FAK) was estimated using the Image J program.

Cell extracts were prepared in lysis buffer (50 mM Tris-HCl pH 6.8, 120 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM β-mercaptoethanol, 1 mM Na₃VO₄, 1 mM NaF, and 1 μg/ml aprotinin) for 30 min on ice, and centrifuged at 15,000 × g for 15 min to remove insoluble materials. Lysates were centrifuged at 14,000 rpm for 10 min, and the resulting supernatants were used for immunoblotting. Protein concentrations were determined using the Pierce protein assay reagent (Pierce, Rockford, IL, USA). Cell lysates containing equal amounts of protein (40 μg) were analyzed by SDS–PAGE. After transferring to Hybond-P membranes and blocking with 5% milk/0.1% TBST at room temperature for 60 min, membranes were incubated first with primary antibodies AR (Millipore PG-21 #06-680), FAK (BD Biosciences #610088), p-FAK(BD Biosciences #611722), ARA5 (BD Biosciences #6111645), Intergrin β1 (Cell Signaling #4706), GAPDH (Millipore #AB2302) overnight at 4 °C, and then with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies. Immunoreactive proteins were detected using an enhanced chemiluminescence detection kit (Millipore). Signals were analyzed and quantified using an LAS-3000 imaging system (Fujifilm, Tokyo, Japan).