Ecl western detection reagent

Cells were lysed using ice-cold lysis buffer (0.1 mL of 50 mM Tris-HCl (pH 7.2) containing 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, and 1% NP-40), and the so-obtained cell lysates were assayed for protein concentration by Bradford staining. Equal amounts of protein (20 μg/mL) were electrophoresed on 10% SDS-acrylamide gels and transferred to nitrocellulose membranes using an electric transfer system. Nonspecific binding was blocked by treating membranes with 3% skim milk in TBST buffer (5 mM Tris-HCl, pH 7.6, 136 mM NaCl, and 0.1% Tween-20) for 1 h. Blots were incubated for 1 h at room temperature with primary antibody against anti-phospho-AMPKα (Thr 172), anti-AMPKα, anti-phospho-acetyl-CoA carboxylase (ACC) (Ser79), anti-ACC, anti-SIRT1 (Cell Signaling Technology, Danvers, MA, USA), anti-PGC1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), glucose transporter (GLUT) 4 (Santa Cruz Biotechnology), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) and then incubated for 1 h at RT with horseradish peroxidase- (HRP-) labeled anti-mouse IgG (1 : 1000, Santa Cruz Biotechnology), washed with 1x TBST three times, and developed with the ECL Western detection reagents (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Protein bands were quantified by densitometry using Image J.

P53 and β-actin were detected by antibodies purchased from commercial sources (mouse monoclonal anti-TP53, clone #2524, Cell Signaling; mouse monoclonal anti-β-actin, EMD Millipore, Massachusetts, USA). Rabbit polyclonal antibodies against AMAP1 were established as described previously [18 (link)]. Peroxidase-conjugated donkey antibodies against mouse or rabbit IgGs were purchased from Jackson ImmunoResearch Laboratories, Inc. All immunoblotting analyses were performed as described previously [16 (link)] using ECL Western detection reagents (GE Healthcare, Illinois, USA).

The mouse monoclonal antibody against β-actin was purchased from a commercial source (EMD Millipore). Rabbit polyclonal antibodies against EPB41L5 were established as described previously [17 (link)]. Peroxidase-conjugated donkey antibodies against mouse or rabbit IgGs were purchased from Jackson ImmunoResearch Laboratories, Inc. Immunoblotting analysis was performed as described previously [6 (link)] using ECL Western detection reagents (GE Healthcare).