Luminol enhancer reagents

Fetal bovine serum (FBS), resveratrol, o-phenylenediamine dihydrochloride (OPD), dimethyl sulfoxide (DMSO), cytochalasin B (CB), and anti-c-myc antibodies were purchased from Sigma Life Sciences (St. Louis, MO, USA). Minimum essential medium (α-MEM), trypsin, and antibiotic-antimycotic (100 U/mL penicillin, 100 µg/mL streptomycin and 250 ng/mL amphotericin B) were purchased from GIBCO Life Technologies (Burlington, Ont., Canada). Antibodies against total IRS-1, phospho-ser³⁰⁷ IRS-1, phospho-ser^636/639 IRS-1, total mTOR, phospho-ser²⁴⁴⁸ mTOR, total p70-S6K, phospho-thr³⁸⁹ p70S6K, total AMPK, phospho-thr¹⁷² AMPK, total Akt, phospho-ser⁴⁷³ Akt, β-actin, and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies were purchased from New England Biolabs (NEB; Mississauga, Ont., Canada). Additionally, peroxidase-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Lab (West Grove, PA, USA). [3H]-2-deoxy-D-glucose was purchased from PerkinElmer (Boston, MA, USA). Insulin (Humulin R) was from Eli Lilly (Indianapolis, IN, USA). Luminol Enhancer reagents, Bradford protein assay reagent, polyvinylidene difluoride (PVDF) membrane, and electrophoresis agents were purchased from BioRad (Hercules, CA, USA).

After treatment, whole lysates were obtained by washing the cells with ice-cold HBS solution and lysing with ice-cold lysis buffer. Lysates were kept at −20 °C. Protein samples (15 µg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane followed by exposure to blocking buffer containing 5% w/v dry milk powder in Tris-buffered saline and overnight incubation with the primary antibody at 4 °C followed by exposure to HRP-conjugated anti-rabbit antibody for 1 h and Luminol Enhancer reagents (BioRad). The corresponding bands were visualized using FluroChem software (ThermoFisher, Waltham, MA, USA).