FIGURE 8.

B-Raf is required for the sustained activation of ERKs by cAMP. A, Hek293 cells were transfected with NS shRNA, or shRNA against B-Raf or C-Raf, and treated with F/I for 0, 2, 5, and 10 min, as indicated. The endogenous levels of pERK and total ERK2, and the efficacies of the shRNAs are shown. B, cells were transfected with NS shRNA or shRNA against B-Raf. Cells were also transfected with FLAG-ERK2 and either vector or HA-tagged shRNA-resistant B-Raf (HA-B-Raf*). Cells were treated with F/I for 10 min, lysed, and subjected to FLAG IP. The levels of ERK activation (pFLAG-ERK2), total FLAG-ERK2 within the IP are shown in the 1st and 2nd panels. The levels of B-Raf within the TCL are shown. Note that both endogenous B-Raf and HA-B-Raf* can be seen as a doublet in the 3rd lane. C, cells were transfected with GFP-ERK2 and NS shRNA or shRNA against C-Raf and either vector or FLAG-tagged shRNA-resistant C-Raf (FLAG-C-Raf*). Cells were treated with EGF for 5 min or left untreated, lysed, and subjected to GFP IP. The levels of activation of GFP-ERK2 (pGFP-ERK2) and total GFP-ERK2 within the IP are shown in the 1st and 2nd panels, respectively. The levels of GFP-ERK2 within the TCL are shown in the 3rd panel. The levels of C-Raf and FLAG-C-Raf* within the TCL are shown in the 4th panel.