FIG. 5.

mTOR participates in the regulation of Bim by PI3-K pathways. (A and B) Immunoblot analysis with Bim antibodies. Cells were cultured in IL-3-free medium for the indicated times. Baf-3 cells containing Dex-inducible Ras(G12V) were cultured with 10⁻⁷ M Dex in the absence (upper panel) or presence (lower panel) of wortmannin (WMN) (0.5 μM) (A). Baf-3 cells containing Dex-inducible Ras(G12V/V45E) were cultured with 10⁻⁷ M Dex (top panel), in the presence of wortmannin (upper middle panel) or rapamycin (Rapa) (10 ng/ml; lower middle panel); Baf-3 cells containing Dex-inducible Ras(G12V/V45E) were engeneered to express an AU1-tagged rapamycin-resistant form of mTOR (AU1-mTOR-rr) (see Materials and Methods) and were cultured with rapamycin (bottom panel) (B). (C) Immunoblot analysis of AU1-tagged mTOR-rr and an endogenous mTOR protein in cell extracts from Baf-3 cells containing Dex-inducible Ras(G12V/V45E) (V45E; lanes 1 and 3) or expressing AU1-mTOR-rr (rr, lanes 2 and 4), using monoclonal antibodies specific for AU1 (lanes 1 and 2) and mTOR (lanes 3 and 4). (D) Percent viability of cells expressing Ras(G12V/V45E), with or without mTOR-rr (rr), in the presence or absence of rapamycin in IL-3-free medium. Also shown is that of cells expressing Ras(G12V/V45E) and MAA-PKB in IL-3-free medium.