Monoclonal anti ampk

Monoclonal anti-α-tubulin antibody was purchased from Santa Cruz Biotechnology, monoclonal anti-AMPK, ERK, mTOR, ACC, p-AMPK, p-ERK, p-mTOR and p-ACC antibodies from Cell Signaling Technology (CST) and monoclonal anti-puromycin antibody from EMD Millipore Co. (Merck Millipore). Penicillin, streptomycin, fetal bovine serum (FBS) and trypsin were purchased from GIBCO Life Technologies Inc., EMEM (LM007-75 and LM007-87 for glucose starvation and normal glucose condition, respectively) and DMEM (LM001-56) media from Welgene Biotech Co. Each media contains same concentrations of ingredients except glucose. Media for glucose-starved condition, low-glucose condition and normal glucose condition contain 0, 500 and 1000 mg/L (0, 2.8 and 5.5 mM) of glucose, respectively.

Cells were washed twice with TBS (10 mM Tris-HCl, pH 7.5, and 150 mM NaCl), lysed in the cell lysis buffer containing 10 mM sodium molybdate, 10 mM sodium fluoride, and 1 mM sodium orthovanadate, sonicated, and centrifuged at 20,000 × g for 10 min. The supernatant was subjected to SDS-PAGE, followed by Western blotting with rabbit monoclonal anti-phospho-AMPK (Thr172) (1/3000, #2535; Cell Signaling Technology, Beverly, MA, USA), monoclonal anti-AMPK (1/3000, #2532; Cell Signaling Technology), rabbit polyclonal anti-phospho-CREB (1/3000, Signalway antibody, College Park, MD, USA), and rabbit monoclonal anti-CREB (1/3000, #9197; Cell Signaling Technology). After incubation with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA), the immunoreactive bands were developed using Immobilon Western substrate (Millipore, Bedford, MA, USA) and read on LAS4000 system (GE Healthcare, Piscataway, NJ, USA). The band intensities were quantified using Image J (ver. 1.44p, National Institutes of Health, Bethesda, MD, USA).