Liver tissue was homogenized as previously described [4 (link),34 (link)]: 50 μg proteins were separated on 6%–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were blotted into polyvinylidene fluoride (PVDF) membranes (Biorad, Madrid, Spain) and immunoblotted overnight at 4 °C with antibodies directed against total and phospho-AKT (Ser473), cleaved caspase 3, cleaved caspase 9, total and phospho-GSK3β (ser 9), cytochrome C, and phospho-VDAC (Cell Signaling, Beverly, MA, USA); GRP78, CHOP, ATF4 and total and phospho-PERK (Santa Cruz Biotechnology, Santa Cruz, California, USA), HMGB-1(Abcam, Milton, Cambridge, UK), and β actin (Sigma Chemical, St. Louis, MO, USA). After washing, membranes were incubated with appropriate peroxidase-conjugated secondary antibody for 1 h. Immuno-labeled bands were detected using Western-Bright ECL-HRP Substrate (Advansta, Menlo Park, CA, USA) and quantified using the Quantity One software for image analysis. Results were expressed as densitometry ratios between the protein of interest and the loading control (β-actin).
Free full text: Click here
