Figure 3.

Effects of TGF-α on PI3K p85, Akt, GSK3-β, and cyclin D1 in all cell lines. (A) C10, A5, E10, and E9 cell lines at 70% confluence were serum starved and treated with TGF-α for 5 min in the presence or absence of inhibitor PD153035. Lysates were immunoprecipitated with anti-EGFR and immunoblotted with anti-p85. A TGF-α–stimulated, PD153035-inhibited complex was detected in all four lines. (B) C10, A5, and E10 cells pretreated with or without the inhibitor PD153035 were stimulated for 5 min with TGF-α. Lysates from these cells were immunoprecipitated with anti-ErbB2 and immunoblotted with anti-p85. (C) The four cell lines were treated as described previously for 5 or 10 min and immunoblotted, with sequential stripping, for EGFR, pEGFR, Akt, and pAkt. EGFR and Akt phosphorylations were observed in all four lines in response to TGF-α and were inhibited by PD153035. (D) The four cell lines were treated as described previously for intervals between 10 min and 6 h with or without the PD153035 inhibitor of EGFR or LY294002 specific for PI3K and immunoblotted for total Akt and pAkt. Total Akt did not change. Akt phosphorylation reached a maximum by 30 min and was markedly reduced by both inhibitors. (E) The four cell lines were treated as described previously for 5 or 10 min in the presence or absence of the PD153035 or LY294002 inhibitors and immunoblotted for phosphorylated GSK3-β. In all lines, the latter was increased by TGF-α treatment, and the increase was partially blocked by each inhibitor. (F) The four cell lines were treated as described previously for intervals of 30 min to 6 h in the presence or absence of PD153035 or LY294002 and immunoblotted for cyclin D1. The latter increased progressively over this time course, and this increase was completely blocked by each inhibitor.