Anti gapdh hrp conjugate

After centrifugation and supernatant discard, the proteins were extracted from the pellet using RIPA Buffer and quantified using Bradford Assay (Bio-Rad, Hercules, CA, USA). After denaturation at 95 °C, 25 µg of protein for each samples underwent separation using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then they were transferred on PVDF membrane (Immobilon–P, Millipore, Burlington, MA, USA). Then, 5% skimmed milk in TBS was used for blocking at room temperature for 1 hour, followed by the overnight incubation at 4 °C. The antibodies used were: Anti-CB1 (1:500, ThermoFisher Scientific, Rockford, IL, USA), anti-GAPDH HRP conjugate (1:1000, Cell Signaling, Danvers, MA, USA), anti-p-Akt (1:1000; Cell Signaling, Danvers, MA, USA), anti-Akt (1:1000, Cell Signaling, Danvers, MA, USA), anti-p-ERK1/2 (1:2000; Cell Signaling, Danvers, MA, USA) and anti-ERK2 (1:1000; Cell Signaling, Danvers, MA, USA). The antibody mouse anti-rabbit IgG-HRP (1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a secondary antibody to be incubated with the membranes for 1 h at room temperature. In order to acquire the bands, ChemiDoc™ MP System (Bio-Rad) was after exposition to enhanced chemiluminescence system (Luminata Western HRP Substrates, Millipore, Burlington, MA, USA).

Following the drug treatment, cells were lysed for 20 min on ice with buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM EDTA 1% Nonidet P-40, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 50 mM β-glycerolophosphate, 1 mM PMSF, 1 mM sodium orthovanadate, and a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). Proteins (50 μg) were separated by SDS-PAGE and transferred to PVDF membranes. The following antibodies were used: anti-PSA (#5877 Cell Signaling Technology, Danvers, MA, USA), anti-AR (#5153S Cell Signaling Technology), anti-GAPDH-HRP conjugate (#8884S Cell Signaling Technology), anti-γH2AX (#05-636-I, Millipore, Burlington, MA, USA), #anti-p53 (DO-I, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p21 (#2947 Cell Signaling Technology), anti-PARP (#9542 Cell Signaling Technology), and anti-β-actin (C4, Santa Cruz Biotechnology, Dallas, TX, USA). Immunoblots were developed using the Li-COR Odyssey imaging system, except for the anti-PSA, anti-AR, and anti-GAPDH-HRP conjugate, which were visualized using an enhanced chemiluminescence detection kit, the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).