P irs ser307

The epididymal adipose tissues of each mouse were homogenized in an extraction buffer containing 100 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM sodium pyrophosphate, 50 mM NaF, 100 mM orthovanadate, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 2 μg/mL aprotinin, 1 μg/mL pepstatin A, and 1 μg/mL leupeptin. The tissue homogenates were centrifuged at 1,300 ×g for 20 min at 4°C. The protein concentrations of the tissue extracts were measured via Bradford assay (Bio-Rad, CA, USA). The protein samples were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (Amersham, Buckinghamshire, UK). The samples were incubated overnight and hybridized with primary antibodies (diluted 1 : 1,000) at 4°C. Antibodies to the following proteins were purchased from the indicated sources: β-catenin, β-actin (Santa Cruz Biotechnology, CA, USA), c-Jun N-terminal kinase (JNK), p-JNK (Tyr183), insulin receptor substrate (IRS), p-IRS (Ser307), protein kinase B (AKT), p-AKT (Thr308), GLUT4, and GAPDH (Cell Signaling Technology, MA, USA). The membranes were incubated with the corresponding secondary antibody. Next, immunoreactive signals were detected via a chemiluminescent detection system (Amersham, Buckinghamshire, UK) and quantified using Quantity One analysis software (Bio-Rad).

The epididymal adipose tissues of each mouse were homogenized in an extraction buffer containing 100 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM sodium pyrophosphate, 50 mM NaF, 100 mM orthovanadate, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 2 μg/mL aprotinin, 1 μg/mL pepstatin A, and 1 μg/mL leupeptin. The tissue homogenates were centrifuged at 1,300 ×g for 20 min at 4°C. The protein concentrations of the tissue extracts were measured via Bradford assay (Bio-Rad, CA, USA). The protein samples were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (Amersham, Buckinghamshire, UK). The samples were incubated overnight and hybridized with primary antibodies (diluted 1 : 1,000) at 4°C. Antibodies to the following proteins were purchased from the indicated sources: β-catenin, β-actin (Santa Cruz Biotechnology, CA, USA), c-Jun N-terminal kinase (JNK), p-JNK (Tyr183), insulin receptor substrate (IRS), p-IRS (Ser307), protein kinase B (AKT), p-AKT (Thr308), GLUT4, and GAPDH (Cell Signaling Technology, MA, USA). The membranes were incubated with the corresponding secondary antibody. Next, immunoreactive signals were detected via a chemiluminescent detection system (Amersham, Buckinghamshire, UK) and quantified using Quantity One analysis software (Bio-Rad).