FIGURE 9.

Ras, Rap1, C-Raf, and B-Raf are all required for cAMP-dependent induction of c-FOS protein. A, Hek293 cells were treated with F/I for the times indicated in the presence and absence of U0126. In one condition, U0126 was applied at 30 min after F/I, and cells were harvested at 60 min. The endogenous levels of c-FOS were detected by Western blotting (1st panel). The levels of pERK and total ERK2 are shown (2nd and 3rd panels). B, cells were transfected with NS shRNA or shRNA against human Ras and Rap1, as indicated, and treated with F/I for 60 min. The levels of c-FOS are shown in the upper panel. The levels of Rap1, KRas, and total Ras are shown in the middle three panels, respectively, Total ERK2 levels are shown as a loading control (bottom panel). C, cells were transfected with NS shRNA, or shRNA against human B-Raf, and C-Raf, as indicated, and treated with F/I for 60 min. The levels of c-FOS are shown in the 1st panel. The levels of B-Raf and C-Raf are shown in the 2nd and 3rd panels, respectively, Total ERK2 levels are shown as a loading control (4th panel). D, shRNA data from three independent experiments representing those shown in B and C are presented as bar graphs, with c-FOS levels calculated by ImageJ densitometry and presented as relative densities normalized to the average densities seen in NS-treated cells. Error bars show standard error. Significance of the differences between densities in each condition and those seen in NS controls was assessed by unpaired Student's t tests (NS = not significant, * = <0.05, and ** = <0.01, and *** = <0.001).