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cAMP agonists inhibit the phosphorylation of Raf-1 on serine 338. COS-1 cells were transfected with pCMV5Raf-1 or pCMV5-RafS259A, serum starved overnight, and treated with 40 μM forskolin plus 50 μM IBMX for 20 min and 100 ng of TPA per ml for 15 min as indicated. (A) The Raf-1 proteins were immunoprecipitated with the crafVI antibody and immunoblotted with anti-phosphoserine 338 and anti-serine 259 antibodies. After being stripped, the blots were stained with crafVI antiserum.
