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. 2005 Apr;25(7):2558–2572. doi: 10.1128/MCB.25.7.2558-2572.2005

FIG. 3.

FIG. 3.

Effects of PI 3-kinase inhibitors or PTEN on the phosphorylation of specific sites in 4E-BP1. (A and B) CHO cells were starved of serum and then, in some cases (−AA), were also starved of amino acids (60 min). Amino acids were then added back to some plates for 30 min (−AA/+AA). Where indicated, cells were treated with LY294002 or wortmannin (WM) (at the indicated concentrations, for 40 min) and then, where indicated, with insulin (Ins). Samples of lysates were analyzed by Western blotting using the indicated antisera for 4E-BP1 or specific phosphorylation sites in 4E-BP1 (A) or for (P)PKB (Thr308) or S6K1 (B), as indicated. (C) Serum-starved HEK293 cells were treated with insulin or with PI 3-kinase inhibitors as described for panels A and B for CHO cells. (D) HEK293 cells were transfected with a vector encoding rat 4E-BP1, plus vectors for wild-type PTEN, the indicated PTEN point mutants, or the empty vector (pcDNA). Samples of lysate (20 μg of protein) were analyzed by SDS-PAGE and Western blotting using antisera for 4E-BP1, specific sites in 4E-BP1, PTEN, or phosphorylated PKB, as indicated.