5 amino 4 imidazole carboxamide riboside aicar

Primary antibodies specific for phospho-AMPKα (172), phospho-HER2 (1221/1222, 1248, 1196), phospho-HER2/phospho-EGFR (1248/1173), phospho-HER3 (1289), phospho-EGFR (1068, 1046/1047S, 1045), phospho-ACC (79), phospho-AMPK substrates (LXRXXpS/pT), AMPKα, AMPKβ1/2, ACC, PARP, HER2, EGFR, HER3, LKB1 and Myc-tag were purchased from Cell Signaling Technology. Anti-rabbit and anti-mouse secondary antibodies were also purchased from Cell Signaling Technology. Anti-phospho-EGFR (1142) was purchased from ECM Biosciences. Anti-Actin antibody was purchased from Sigma-Aldrich. 5-Amino-4-imidazole carboxamide riboside (AICAR), a pharmacological activator of AMPK (Toronto Research Chemicals, Toronto, ON) was dissolved in DMSO to make a 500mM stock concentration. EGF (Sigma) was prepared in a stock concentration of 100μg/ml in purified water. Compound C, an inhibitor of AMPK, was obtained from Calbiochem, and G418, used for selection of transfected cells, was obtained from Invitrogen. Control, AMPKα1/2, AMPKβ1, and AMPKβ2 siRNAs were purchased from Santa Cruz Biotechnology. All siRNA transfections were conducted in triplicate over a period of 72 hours using RNAiMax (Invitrogen) according to the manufacturer's recommendation.