PKB activity in IRS-1–deficient primary hepatocytes and liver. (a) The effect of IRS-1wt or IRS-1Δp85 on PKB phosphorylation and activity in IRS-1–deficient primary hepatocytes. After being treated without or with 100 nM insulin for 5 minutes, the cell lysates were subjected to SDS-PAGE, followed by Western blot analysis (IB) with anti-PKB antibodies (αPKB-CT) (upper panel), or subjected to immunoprecipitation (IP) with αPKB-CT followed by the immune complex kinase assay as described in Methods (lower panel). The results are expressed as the ratio to the value of LacZ without insulin, and each bar represents the mean ± SD of more than three independent experiments. ^AP < 0.05 LacZ insulin (+) versus IRS-1wt insulin (+). ^BP < 0.05 LacZ insulin (+) versus IRS-1Δp85 insulin (+). (b) The effect of IRS-1wt or IRS-1Δp85 on PKB activity in IRS-1–deficient mouse liver. After being treated without or with insulin for 5 minutes, the liver lysates were immunoprecipitated with αPKB-CT and then subjected to PKB kinase assay. The results are expressed as the ratio to the value of LacZ without insulin, and each bar represents the mean ± SD of more than three independent experiments. ^AP < 0.01 LacZ insulin (+) versus wild-type insulin (+). ^BP < 0.05 LacZ insulin (+) versus IRS-1wt insulin (+). ^CP < 0.05 LacZ insulin (+) versus IRS-1Δp85 insulin (+). (c) The effect of PI3K inhibition on PKB activation on IRS-1 deficient primary hepatocytes. The indicated proteins were introduced into IRS-1–deficient primary hepatocytes by adenovirus-mediated gene transfer. Cells were incubated with or without 50 nM wortmannin for 30 minutes and were treated with 100 nM insulin for 5 minutes. The cell lysates were subjected to immunoprecipitation with 4G10 or αPKB-CT, followed by PI3K assay (upper panel) and PKB immune complex kinase assay (lower panel), respectively. In the lower panel, the results are expressed as the ratio to the value of LacZ without insulin, and each bar represents the mean of two experiments. wor, wortmannin.