P irs 1 tyr632

Mature adipocytes were treated with PPE for 24 h. Medium was removed and replaced with DMEM containing 10 nM insulin for 30 min. After washing with PBS, cells were collected in lysis buffer, followed by centrifugation at 13,000 rpm for 20 min to collect the supernatant. Protein concentrations were determined by the Bradford (Sigma, St. Louis, MO, USA) method and equal amounts of protein (25 µg for each) were separated in 10% SDS-PAGE gels and transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membrane was blocked for 1 h at 4℃ with 5% nonfat milk in TBS-T buffer [10 mM Tris-HCl (pH 6.8), 100 mM NaCl, and 0.1% Tween 20]. The membranes were then incubated with primary antibodies [p-IRS-1 (Ser³⁰⁷) 1:1000, IRS-1 1:1000, Akt 1:1000, p-Akt (Ser⁴⁷³) 1:1000, and GLUT4 1:1000; Cell Signaling Technology, Beverly, MA, USA; p-IRS-1 (Tyr⁶³²) 1:1000 and β-actin 1:1000; Santa Cruz Biotechnology Inc., Dallas, Tex, USA] overnight at 4℃ and then with horseradish peroxidase conjugated secondary antibody [goat anti-rabbit or goat anti-mouse (1:2000), Jackson ImmunoResearch Inc, West Grove, PA, USA] for 1 h. Bands were detected by chemiluminescent substrate (IMGENEX ,San Diego, CA, USA), and the protein expression was quantified using UVP (imaging system for chemiluminescent Western blot, Upland, CA, USA) and Vision Works TMLS (Analysis Software, Upland, CA, USA).