Anti pampkα1 thr172

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).

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).

Capsaicin (CAP), AICAR, dorsomorphin (also named compound C), troglitazone and GW9662 were obtained from Tocris (Ellisville, MO, USA). BODIPY (493/503) and STO-609 (a CaMKKβ inhibitor) were from Sigma (St. Louis, MO, USA). Anti-pAMPKα1-Thr172, anti-AMPK, anti-pACC-Ser79, anti-ACC, anti-pSREBP-1c-Ser372, anti-pAKT-Ser473, anti-AKT, anti-pmTOR-Ser2248, anti-mTOR, anti-pS6-Thr389, anti-S6, anti-p62 from Cell Signaling Technology (Danvers, MA, USA), anti-SREBP, anti-CD36, anti-PPARα from Abcam (Cambridge, UK), anti-PPARγ from Santa Cruz Biotechnology (Dallas, TX, USA), anti-PGC-1α, anti-LC3B from Novus Biologicals (Abingdon, UK) and anti-β-tubulin from Sigma (St. Louis, MO, USA).