Western Blotting Analysis of miR-214 and PTK6 in Prostate Cancer

Western blotting was performed as described previously^{71 (link)}. After transfection with miR-214 or NC mimic for 48 h, cell lysates were prepared from RWPE-2, PC3, DU145, MDA-PCa-2b, and LNCaP cells using lysis buffer (Cell Signaling Technology, Danvers, MA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN). Cell lysates were also prepared from RWPE-2, PC3, DU145, and MDA-PCa-2b cells after transfection with empty vector or PTK6 plasmid for 24 h. Protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). Cell lysates (40 µg) were separated on NuPAGE 4–12% Bis-Tris-SDS gels (Invitrogen) and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membrane was blocked in casein blocking buffer (1×) (Sigma-Aldrich, St. Louis, MO) and incubated with primary antibodies against cleaved PARP, PTK6, E-Cadherin, N-Cadherin, and GAPDH (Cell Signaling Technology) overnight at 4 °C. The following day, membranes were washed and subsequently incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Following this incubation, membranes were washed and developed with an enhanced chemiluminescent detection system (Thermo Fisher Scientific, Waltham, MA).