WT2 (panel A) and V7 (panel B) cells were incubated for the indicated times with 100 nM PMA. Whole-cell extracts, equalized for protein, were immunoblotted for phospho-Raf (Ser259, Ser289/296/301, Ser338), c-Raf, phospho-Erk, and actin. In panel C, WT2 and V7 cells were treated with PMA for 3 hours. Whole-cell extracts, equalized for protein, were run on the same gel. Blots were probed for phospho-Raf (Ser259, Ser289/296/301, Ser338), and c-Raf to show the physical location of multiple Raf species. All incubations were done in the same experiment; the blots were exposed in parallel.