Anti rabbit and anti mouse secondary antibody

Western blotting analysis was performed as described previously [26 ] using the following detection antibodies: anti-rabbit RBM38 (Santa Cruz, USA), anti-mouse c-Myc (Thermo, USA), anti-mouse β-actin (Cell Signaling technology, USA) and anti-rabbit and anti-mouse secondary antibodies (Cell Signaling technology, USA).

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.

Cells were lysed with RIPA buffer (Thermo Fisher Scientific) in the presence of a protease inhibitor cocktail (Thermo Fisher Scientific) and sonicated for 30 seconds. Lysates were separated by SDS-PAGE under reducing conditions, transferred to a PVDF membrane, and analyzed by immunoblotting. Rabbit anti-CPT1A (No. 12252) (Cell Signaling Technology) was used as a primary antibody. Immunoblotting for β-tubulin by mouse anti-β-tubulin antibody (Santa Cruz Biotechnology) and COX IV by rabbit anti-COX IV antibody (Cell Signaling) was used as a loading control for whole-cell lysates and mitochondrial lysates, respectively. Anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling Technology and Thermo Fisher Scientific, respectively. Signal was detected using the ECL system with X-ray film development (Thermo Fisher Scientific and GE Healthcare Life Sciences) or a LI-COR C-Digit blot scanner (LI-COR) according to the manufacturer’s instructions.

I3C, purchased from Sigma-Aldrich (St. Louis, MO, USA), was dissolved in Dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) and 400 mM stock solutions of this preparation were stored at -20°C. EZ-Cytox was purchased from DoGenBio (Seoul, Korea). Caspase-3, caspase-7, caspase-9, and cleaved Caspase-3, caspase-7, caspase-9, PARP, cleaved PARP, Akt, pAkt, Bcl-xL, Bim, Bad, Fas, and β-actin primary antibodies were obtained from Cell Signaling Technology (Danvers, MA). FOXO3, caspase-8, ERK, pERK, JNK, pJNK, p38, and pp38 primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-rabbit and anti-mouse secondary antibodies were purchased from Cell Signaling Technology.

TLR4 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for P38 MAPK, SAPK/JNK, ERK, and MyD88, and anti-rabbit and anti-mouse secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA). Pg-LPS was purchased from in vivo Gen (San Diego, CA, USA).