Technical Reference #1733

1733.

Hepatic Autophagy Is Suppressed in the Presence of Insulin Resistance and Hyperinsulinemia: Inhibition of Fox01-Dependent Expression of Key Autophagy Genes by Insulin Hui-Yu Liu; Jianmin Han; Sophia Y. Cao; Tao Hong; Degen Zhuo; Jianbo Shi; Zhenqi Liu; Wenhong Cao, Hamner Institutes for Health Sciences Sun Yat-sen University University of Virginia Health System Duke University Health System, Journal of Biological Chemistry, 284(1733), (2009)
Link To Paper

Materials & Methods:
Antibodies against LC3 and p62 were from MBL International (Woburn MA). The monoclonal antibody against -actin the antibody against ubiquitin the recombinant human insulin and glucagon were from Sigma.Antibodies against total and phospho-Akt (serine 473/308) or phospho-FoxO1 were from Cell Signaling Technology (Danvers MA). Antibodies against IRS1(Tyr(P)416) was from Abcam (catalog number ab66153). Antibodies against PARP1 were from Santa Cruz Biotechnology (catalog number SC-7150). LY294002 was from Calbiochem. Blood glucose concentrations were measured using a Breese 2 glucose meter (Bayer Health- Care). Plasma insulin levels were measured with a Linco insulin enzyme-linked immunosorbent assay kit. Protein assay kits were from Bio-Rad. The plasmid LC3-GFP and the recombinant adenoviruses encoding a constitutively nuclear FoxO1 mutant (ADA-FoxO1) were kind gifts from Dr. Tamotsu Yoshimori (Osaka University Osaka Japan) and Dr. Domenico Accili (Columbia University New York NY) respectively. Other materials were all obtained commercially and are of analytical quality

Microscopic Technique
Confocal Microscopy

Cell Type(s)
Mouse primary hepatocytes (Hepatoma 1c1c7 cells)