Las 3000 gel documentation system

Total cellular proteins were extracted from cultured BMMs or OCs using RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate) supplemented with Protease Inhibitor Cocktail (Roche). Lysates were cleared by centrifugation at 16,000 g at 4°C for 20 mins and supernatants containing proteins were collected. For immunoblotting, 30μg of extracted proteins diluted in SDS-sampling buffer was resolved by SDS-PAGE (10–15%) gels and then electroblotted onto nitrocellulose membranes (Hybond ECL, Amersham Life Science). Following transfer, membranes were blocked with 5% (w/v) skim milk in TBS-Tween (TBS; 0.05 M Tris, 0.15 M NaCl, pH 7.5 and 0.1% Tween-20) for 1 hr and then probed with primary antibodies diluted in 1% (w/v) skim milk powder in TBS-Tween at 4°C overnight. Membranes were washed and then incubated with HRP-conjugated secondary antibodies and antibody reactivity was detected by the Western Lightning Ultra Detection Kit (PerkinElmer, Waltham, MA, USA) using the FujiFilm LAS-3000 Gel Documentation System (FujiFilm, Tokyo, Japan) and its associated software.

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.