The effect of IRS-1 reconstitution by adenovirus-mediated gene transfer in the IRS-1–deficient mice. The IRS-1–deficient mice were treated with the adenoviruses as described in Methods. (a) Specific reconstitution of IRS-1 protein in the knockout mouse liver. The lysates of each tissue (liver, soleus muscle [muscle], and epididymal fat pad [fat]) from the indicated mouse (wild-type mouse [wild], null mouse treated with LacZ adenovirus [null LacZ], and null mouse treated with wild-type IRS-1 [null IRS-1wt]) were prepared as described in Methods. The lysates were immunoprecipitated (IP) with the antibodies against the COOH-terminal region of IRS-1 (αIRS-1-CT) and subjected to SDS-PAGE (7% gel), followed by Western blotting (IB) with the same antibodies. (b) Tyrosine-phosphorylated proteins in liver in response to insulin. Mice were anesthetized, and 1 μg/g body weight of insulin was injected through the inferior vena cava. After 5 minutes, the liver was removed and lysed with the tissue homogenization buffer as described in Methods. The lysates were immunoprecipitated with anti-phosphotyrosine antibodies (4G10) and subjected to SDS-PAGE (7% gel), followed by Western blotting with the same antibodies. PY, anti-phosphotyrosine antibodies. (c) ITT on mice treated with the indicated adenoviruses. ITT was performed on the indicated group of mice (eight mice in each group). Each bar represents ± SD. ^AP < 0.02 wild-type or null IRS-1wt versus null or null LacZ. ^BP < 0.01 wild-type or null IRS-1wt versus null or null LacZ. ^CP < 0.01 wild-type or null IRS-1wt versus null or null LacZ. (d) Insulin-induced MAP kinase activity in the liver infected with the indicated adenoviruses. After being treated without or with insulin for 5 minutes, the liver lysates were immunoprecipitated with anti-MAP kinase antibodies (αC92) and subjected to the immune complex kinase assay as described in Methods. The results are expressed as the ratio to the value of wild-type without insulin. Each bar represents the mean ± SD of more than three independent experiments. (e) Insulin-induced PI3K activity associated with the tyrosine-phosphorylated proteins in the liver infected with the indicated adenoviruses. After being treated without or with insulin for 5 minutes, the liver lysates were immunoprecipitated with anti-phosphotyrosine antibodies and then subjected to PI3K assay as described in Methods. The lower panel shows the representative results (lanes 1–3; wild-type, lanes 4–6; null LacZ, lanes 7–9; null treated with IRS-1wt, lanes 1, 4, and 7; insulin [–], lanes 2, 3, 5, 6, 8, and 9; insulin [+]). In the upper panel, the results are expressed as the ratio to the value of wild-type without insulin, and each bar represents the mean ± SD of more than three independent experiments. ^AP < 0.05 wild-type insulin (+) versus null LacZ insulin (+). ^BP < 0.05 null IRS-1wt insulin (+) versus null LacZ insulin (+).