Figure 2. Unimpaired insulin signalling through PKB in hepatocytes overexpressing TRB3/NIPK.

Hepatocytes were placed in culture and transduced after 5 h with the AdEasy-1 HA–TRB3/NIPK adenovirus at an MOI of 300 or left uninfected. Cell culture was continued for 18 h after transduction. Insulin was then added (or not added for control cells) to culture medium at a concentration of 3×10⁻⁸ M for 60 min before cell harvest for the extraction of protein, SDS/PAGE and immunoblot analysis of phosphorylated and total PKB, phosphorylated and total GSK3 and total 4E-BP1. Protein load per lane was 40 μg for P-PKB Ser-473, 60 μg for P-PKB Thr-308 and total PKB, and 70 μg for P-GSK3, total GSK3 and 4E-BP1. The experiment was repeated twice with identical results. Positions of M_r markers are indicated on the right.