Persistently activated RasC alters the time course of PKBR1 and PKB substrate phosphorylation. (A) Flag-RasC or -RasCQ62L was expressed under the control of a doxycycline-inducible promoter in rasC− cells. Cells developed in the presence or absence of doxycycline were stimulated with cAMP, sampled at the indicated time points, and probed with phospho-specific antibodies or anti-Flag antibody. The protein-transferred membrane was stained with Coomassie brilliant blue (CBB) and shown as loading control. (B) Quantitative densitometry of the first and second panels of A, showing the mean intensity ± SD of the respective bands from four independent experiments. (C) Samples were probed with anti–phospho-substrate antibody (top). Densitometric scans of the 240-s lanes are shown in bottom panel (Flag-RasC, gray line; Flag-RasCQ62L, black line).
