Western Blot Analysis of Insulin Signaling

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.

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Shu Y., Hassan F., Ostrowski M.C, & Mehta K.D. (2021). Role of hepatic PKCβ in nutritional regulation of hepatic glycogen synthesis. JCI Insight, 6(19), e149023.

Publication 2021

P-AKTThr308 P-AKTSer473 FoxO1 FoxO3a P-FoxO1Ser256 GSK3α/β P-GSK3βSer9 P-GSSer641 P-IRS-1Ser307 P-IRS-1Ser612 P-ERKThr202/Tyr204 HNF-4α PGC-1α β-actin goat anti–rabbit HRP–conjugated secondary antibodies

Actin Antibodies Frozen Gels Goat Gsk3α Immunoblots Mouse Mtor Nitrogen Pgc 1α Proteins Rabbit Sds page Tissue Tsc2

Corresponding Organization : The Ohio State University Wexner Medical Center

Other organizations : MUSC Hollings Cancer Center, Medical University of South Carolina

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