Figure 7.

Effects of IRS proteins on phosphorylation of Bcl-2. (A) Insulin induces phosphorylation of Bcl-2 but not the Δloop mutant of Bcl-2. Left panel, total cell lysates from Bcl-2 and Δloop cells were subjected to immunoblotting with anti-Bcl-2 antibody (N-19). Right panel, Bcl-2 and Δloop cells were serum starved and labeled with [³²P]orthophosphoric acid. Then cells were stimulated with or without insulin (100 nM), lysed, immunoprecipitated with anti-Bcl-2 antibody, and subjected to SDS-PAGE. The phosphorylated proteins were visualized by autoradiography. Δloop, deletion mutant of Bcl-2 lacking the loop domain. (B) IRS-1 but not IRS-3 suppresses insulin-induced phosphorylation of Bcl-2. Bcl-2, IRS-1/Bcl-2, and IRS-3-HA/Bcl-2 cells were serum starved and labeled with [³²P]orthophosphoric acid. Phosphorylation of Bcl-2 was detected as in A. The expression levels of IRS-1, IRS-3, and Bcl-2 were indicated by immunoblotting with the indicated antibodies. (C) Survival rate of double stable transfectants of Ba/F3 cells established by using retrovirus vectors carrying IRS-1-HA-IRES-Δloop and IRS-3-HA-IRES-Δloop, compared with those of Δloop and IRS-3 cells. Δloop, deletion mutant of Bcl-2 lacking the loop domain.