FIGURE 6.

Keratins are sumoylated in primary mouse hepatocytes and in human hepatoma HepG2 cells under oxidative stress, apoptosis, and/or phosphatase inhibition. A, primary hepatocytes from human K18-overexpressing mice were isolated and cultured as described under “Experimental Procedures.” K8/K18 were immunoprecipitated using mouse monoclonal DC10 antibody (anti-human K18), and the immunoprecipitates were resolved, under nonreducing conditions, on a SDS-PAGE gel, which was stained by Coomassie to verify the presence of keratins. Both keratin monomers (mono.) and likely dimers are visible. Immunoblots of the immunoprecipitates verify the presence of keratins, in equal amounts across treatment groups, as detected by rabbit antibodies to human K18 and K8/K18. Immunoblot for SUMO-2/3 (under reducing conditions) shows sumoylated keratins (arrows) in the presence of H₂O₂, OA, and anisomycin (AN) for 6 or 24 h, but not in control hepatocytes. B, untreated (control) and OA-treated HepG2 cells were stained for K18 (DC10 antibody) and SUMO-2/3 followed by Alexa 488 (green) and Alexa 594 (red)-conjugated secondary antibodies and imaged by confocal microscopy. The arrows point to significant colocalization (yellow signal) of keratins and SUMO-2/3 in the presence of OA.