Anti actin monoclonal hrp antibody clone ac15

Western blotting description has already been published elsewhere^{19 (link)}. For transfection of LNCaP and DU145 cells, 6 × 10⁵ cells were cultivated in 6-well plates. After 24 h, cells were transfected with 2 μg of expression plasmid DNA (pSG5-miR-106b, pSG5-RBMS1 or empty pSG5) or 10 nM siRNA (si-RBMS1 or scrambled siRNA) using jetPRIME transfection reagent (Polyplus transfection, Sélestat, France). Cells were lysed 48 h after transfection with 2 × sample buffer (130 mmol/l Tris/HCl, 6% SDS, 10% 3-mercapto-1,2-propandiol, 10% glycerol, and 0.05% bromophenol blue). 30 μg of extracted proteins were separated by 9% Tricine-SDS–Polyacrylamide-Gel electrophoresis and transferred to a nitrocellulose membrane (Whatman, GE Healthcare, Freiburg, Germany) by electroblotting. For immune detection, the primary anti-RBMS1 monoclonal mouse antibody (clone OTI2H1, Thermo Scientific, Schwerte, Germany) and anti-ß-actin monoclonal HRP antibody (clone AC15, Sigma-Aldrich, Hamburg, Germany) were used. Secondary goat anti-mouse HRP clone 31430 antibody was purchased from Pierce (Thermo Scientific, Schwerte, Germany). Bands were visualized by ECL plus Western Blotting Substrate from Pierce (Thermo Scientific, Schwerte, Germany) and the Fujifilm LAS-3000 gel documentation system (Kleve, Germany).

For transfection of LNCaP cells, 6×10⁵ cells were cultivated in 6-well plates. After 24 h, cells were transfected with 2 μg of expression plasmid DNA using jetPRIME^® transfection reagent (Polyplus transfection, Sélestat, France). Cells were lysed 48 h after transfection with 2× sample buffer (130 mmol/l Tris/HCl, 6% SDS, 10% 3-mercapto-1,2-propandiol, 10% glycerol, and 0.05% bromophenol blue). 30 μg of extracted proteins were separated by 9% Tricine-SDS-Polyacrylamide-Gel electrophoresis and transferred to a nitrocellulose membrane (Whatman, GE Healthcare, Freiburg, Germany) by electroblotting. For immune detection the primary antibodies anti-CCND1 monoclonal rabbit antibody (clone SP4, Sigma-Aldrich, Hamburg, Germany) and anti-ß-actin monoclonal HRP antibody (clone AC15, Sigma-Aldrich, Hamburg, Germany) were used. Secondary goat anti-rabbit HRP clone 31460 antibody was purchased from Pierce (Thermo Fisher Scientific, Schwerte, Germany). Bands were visualized by ECL plus Western Blotting Substrate from Pierce (Thermo Fisher Scientific, Schwerte, Germany) and the Fujifilm LAS-3000 gel documentation system (Kleve, Germany) by densitometrical quantification.