FIG. 7.

TSH and Rap1 activity regulate Akt phosphorylation. (A) Cells arrested in basal medium for 48 h were pretreated with H89 and then stimulated with TSH for 45 min. Cell lysates were subjected to Western blotting with an antibody to active phosphorylated (Ser473) Akt1 (Akt-P). Similar results were obtained in three Rap1A63E cell lines in six experiments. (B) Lysates prepared from cells arrested as described above and treated with TSH for the times indicated (in minutes) were analyzed by Western blotting with the phospho-specific Akt antibody. The expression of cellular or mutant Rap1A constructs had no effect on the total levels of Akt1 expression (data not shown). Three experiments in three independent Rap1A63E cell lines were performed with similar results. (C) Cells in basal medium were pretreated with wortmannin, stimulated with TSH for 45 min, and subjected to Western blotting with the phospho-specific Akt1 antibody. Equal protein loading in panels A, B, and C was confirmed by Western blotting with an anti-MAPK2 antibody (data not shown). Three experiments using three independent Rap1A and Rap1A63E cell lines were performed with similar results.