Peroxidase labeled secondary anti mouse igg

Capsaicin (CAP) and Ddocetaxel (DTX) were purchased to TOCRIS (Bristol, UK). dorsomorphin and STO-609 were purchased to Sigma (St. Louis, USA). Primary antibodies anti-pAMPKα1-thr172, pACC-ser79, pAkt-ser473, pmTOR, pS6, pLKB1 and the antibodies against the corresponding total forms were obtained from Cell Signaling Technology (Danvers, MA, USA). Peroxidase labeled secondary anti-mouse IgG was from Sigma (St. Louis, MO, USA) and anti-rabbit IgG was from Calbiochem (San Diego, USA).

Capsaicin (CAP) and Sorafenib were purchased to Sigma (St. Louis, MO, USA). Primary antibodies anti-caspase-9, anti-PARP, anti-AFP, anti-pAkt-ser473, p-mTOR-ser2448, p-AMPKα1-thr172, p-ACC-ser79 and the antibodies against the corresponding total forms were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-caspase-3 antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). (Peroxidase labeled secondary anti-mouse IgG was from Sigma (St. Louis, MO, USA) and anti-rabbit IgG was from Calbiochem (San Diego, CA, USA).

Proteins for Western blotting were isolated by lysing cells in lysis buffer (50 mM Tris pH 7.4, 0.8 M NaCl, 5 mM MgCl2, 0.1% Triton X-100) containing protease inhibitor and a phosphatase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), incubated on ice for 15 min and cleared by microcentrifugation. Twenty micrograms of total protein/lane were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane. The membranes were incubated overnight at 4 °C with primary antibodies. After washing in T-TBS, the membranes were incubated with peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000) for 2 h at room temperature. The immune complex was visualized with an ECL system (Cell Signaling Technology, Danvers, MA, USA). Protein expression levels were quantified using Image J (National Institutes of Health, Bethesda, MD USA) and were expressed as fold changes relative to the control treatment. The primary antibodies (anti-p-AMPKα1-thr172, p-ACC-ser79 and pLKB1-ser428) and the antibodies against the corresponding total forms were obtained from Cell Signaling Technology (Danvers, MA, USA). TRPV1 was obtained from Thermo Scientific (Waltham, MA, USA). Peroxidase-labeled secondary anti-mouse IgG was from Sigma-Aldrich (St. Louis, MO, USA) and anti-rabbit IgG was from Cell Signaling Technology (Danvers, MA, USA).