Total ampkα 23a3

INS-1 cells, C2C12 myotubes, skeletal muscle and interscapular brown fat tissue were mechanically homogenized in ice-cold lysis buffer and centrifuged at 14,000g for 10 min at 4 °C. The resulting supernatant was separated by SDS–PAGE, and immunoblotted with antibodies to sarcomeric myosin heavy chain (MF20; 1:1,000 dilution; Developmental Studies Hybridoma Bank, University of Iowa), DJ-1 (D-4; 1:500 dilution), UCP1 (M-17; 1:500 dilution), actin (C-2; 1:1,000 dilution) (Santa Cruz Biotechnology), phospho-AMPKα (Thr172) (40H9; 1:1,000 dilution), total AMPKα (23A3; 1:1,000 dilution), phospho-Akt (Ser473) (1:500 dilution), total Akt (1;1,000 dilution), Bcl-xL (H-5; 1:500 dilution), GAPDH (14C10; 1:5,000 dilution) or α/β-tubulin (1:1,000 dilution) (Cell Signalling Technology). Protein band intensity was quantified by ImageJ software.

In vitro and ex vivo macrophages (approximately 1×10⁶) and L.infantum promastigotes (1×10⁷), were lysed in ice-cold lysis buffer containing 50 mM Tris, pH 7.4, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 25 mM sodium-β-glycerophosphate, 1 mM DTT, 0.5 mM PMSF, and protease inhibitors (Complete Protease Inhibitor Cocktail; Roche), for 30 min at 4°C. The lysates (twenty to fifty micrograms of protein) were subjected to SDS-PAGE electrophoresis and the proteins were transferred to mini nitrocellulose membranes (Biorad) by the Trans Blot Turbo Transfer System (Biorad). The membranes were then incubated with primary antibodies and with horseradish peroxidase-coupled secondary reagents (Jackson ImmunoResearch). The membranes development was made by Super Signal West Pico or West Dura chemiluminescence substrate (Thermo Scientific). Primary antibodies were directed against: total AMPKα (23A3), AMPKα phosphorylated at Thr172 (#2531; both from Cell Signaling), total SIRT1 (H-300) and total PGC1β (E-9; both from Santa Cruz), total PGC1α (4C1.3; Merck Millipore), and β-actin (C4; Antibodies-online).