Figure 3.

Dominant-negative Akt blocks TGFβ-induced FKHRL1 phosphorylation and Forkhead-dependent transcription but not phosphorylation of Smad2. (A) Mock-transfected and dn Akt-transfected HaCaT cells were incubated in medium with or without 2 μg/ml Tet for 48 h. For the last 24 h of incubation, medium was replaced by serum-free medium ± Tet. The cells were then treated with 2 ng/ml TGFβ for 30 min, harvested, and lysed. Protein extracts (30 μg/lane) were subjected to SDS-PAGE followed by immunoblot as described in MATERIALS AND METHODS. Dilution ratios for primary antibodies are 1:500 for P-Ser473 Akt, FKHRL1, and P-Ser253 FKHRL1, and 1:1000 for total Akt. (B) HaCaT cells were transfected with WT- or TM-FKHRL1, each with FHRE-Luc and pCMV-Rl vectors, and incubated in medium with or without 2 μg/ml Tet for 48 h. For the last 24 h where indicated, medium was replaced with serum-free DMEM ± Tet with or without 2 ng/ml TGFβ. Dual luciferase assay was performed as described in MATERIALS AND METHODS. Each data point represents mean ± SD of four wells. (C) In the presence or absence of Tet, cells were incubated with 2 ng/ml TGFβ for 30 min and lysed as in Figure 3A. Cell lysates were resolved by SDS-PAGE and subjected to immunoblot procedures for total and phosphorylated Smad2. Each lane contains 50 μg of protein. Dilution ratios for primary antibodies are 1:1000 for total Smad2 and 1:500 for P-Smad2.