Western blot analysis

Frozen prostate tissues were homogenized in a buffer containing 25 mM Tris/HCl, 10 μM PMSF, 1 mM benzamidine and 10 μg·mL⁻¹ leupeptine hemisulfate, using the FastPrep®‐24 system with matrix A (MP Biomedicals, Illkirch, France). After centrifugation (20 000 g, 4 min), supernatants were assayed for protein concentration using the Dc‐Assay kit (Biorad, Munich, Germany) and boiled for 10 min with SDS sample buffer (Roth, Karlsruhe, Germany). Samples of WPMY‐1, SYF‐2459, or SYF‐2498 cells were prepared as described below. Samples of prostate homogenates (20 μg per lane) or WPMY‐1 cells (40 μg per lane) were subjected to SDS‐PAGE, and proteins were blotted on Protran® nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were blocked with PBS containing 5% milk powder (Roth, Karlsruhe, Germany) overnight, and incubated with rabbit anti phospho‐Src family (Tyr⁴¹⁶) (#6943) (reacts with different SFK members, including c‐Src, Lyn, Fyn, Lck, Yes, Hck, when phosphorylated at equivalent sites) (New England Biolabs, Ipswich, MA, USA), rabbit anti c‐Src (#2110) (New England Biolabs, Ipswich, MA, USA), rabbit anti Lck (sc‐13), rabbit anti Fyn (sc‐16), rabbit anti Lyn (sc‐15), rabbit anti Fgr (sc‐17), rabbit anti Hck (sc‐72), goat anti Rak (sc‐6377), rabbit anti Yes (sc‐14), rabbit anti Blk (sc‐938), mouse anti pan‐cytokeratin (sc‐8018), mouse anti calponin 1/2/3 (sc‐136987), mouse anti prostate‐specific antigen (PSA) (sc‐7316) or mouse anti β‐actin antibody (sc‐47778) (if not stated otherwise, all compounds were from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, membranes were washed with PBS containing 0.1% Tween 20 (PBS‐T), incubated with secondary biotinylated goat anti rabbit, horse anti mouse or horse anti goat IgG (BA‐1000, BA‐2000, BA‐9500) (Vector Laboratories, Burlingame, CA, USA), washed again with PBS‐T, incubated with avidin and biotinylated horseradish peroxidase from the ‘Vectastain ABC kit’ (Vector Laboratories, Burlingame, CA, USA) both diluted 1:200 in PBS, and washed again with PBS‐T. Finally, blots were developed with enhanced chemiluminescence (ECL) using ECL Hyperfilm (GE Healthcare, Freiburg, Germany).