P foxo1 ser256

INS-1E cells treated with leptin (10 nM) or/and TNF-α (20 ng/ml) were collected, and total protein extracts were prepared as previously described [25 (link)]. Briefly, 50 μl of total protein sample was subjected to 12% SDS-PAGE and transferred overnight at 4°C onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with blocking buffer (20 mM Tris, 150 mM NaCl, pH 7.5, and 5% nonfat dry milk) at room temperature for 1 hour and then incubated with primary antibodies against p-STAT3 (1 : 500, Santa Cruz Biotechnology, CA, USA, sc-8059), STAT3 (1 : 1000, Santa Cruz Biotechnology, CA, USA, sc-8019), LepRb (1 : 400, Santa Cruz Biotechnology, CA, USA, sc-8325), p-FOXO1[Ser²⁵⁶](1 : 1000, Cell Signaling Technology, MA, USA, 9461S), FOXO1 (1 : 1000, Cell Signaling Technology, MA, USA, 9454S), SOCS3 (1 : 500, Santa Cruz Biotechnology, CA, USA, sc-73045), and β-actin (1 : 1000, Santa Cruz Biotechnology, CA, USA, sc-47778). The antigen-antibody complex was then detected by incubating the membranes for 1 hour in a buffer containing a 1 : 5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (Millipore, Billerica, MA, USA).

The tissues were isolated from mice in the fed condition and then immediately frozen in liquid nitrogen. Tissue homogenates were prepared, and 30–50 μg of protein was resolved on 4%–12% SDS-PAGE gels and subjected to Western blotting (12 (link)). Immunoblots were performed using antibodies against the following proteins: AKT (catalog 4685), P-AKT^Thr308 (catalog 13038), P-AKT^Ser473 (catalog 4060), FoxO1 (catalog 2880), FoxO3a (catalog D19A7), P-FoxO1^Ser256 (catalog 9461), P-FoxO1^Thr24/FoxO3a^Thr32/FoxO4^Thr28 (catalog 2599), FoxO1 (catalog 2880), GSK3α/β (catalog 5676), P-GSK3β^Ser9 (catalog 5558), GS (catalog 3893), P-GS^Ser641 (catalog 94905), P-IRS-1^Ser307 (catalog 2381), P-IRS-1^Ser612 (catalog 3203), P-TSC2^Ser939 (catalog 3615), TSC2 (catalog 3612), p-mTOR^Ser2449 (catalog 5536), mTOR (catalog 2983), and P-ERK^{Thr202/Tyr204} (catalog 9101) were purchased from Cell Signaling Technology. Antibodies against HNF-4α (catalog A2085), PGC-1α (catalog A11971), GK (catalog Ab293), G6Pase (catalog A16234), GKCR (catalog A5678), and β-actin (catalog AC026) were purchased from ABclonal, and ERK (catalog 514302) was purchased from Santa Cruz Biotechnology. Goat anti-mouse and goat anti–rabbit HRP–conjugated secondary antibodies (1:3000; Bio-Rad, 1662408EDU) were used for described experiments.