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. 2002 May;22(10):3237–3246. doi: 10.1128/MCB.22.10.3237-3246.2002

FIG. 2.

FIG. 2.

The role of Raf-1 serine 43. (A) cAMP agonists and TPA induce phosphorylation of S43. COS-1 cells were transfected with pCMV5-Raf-1, serum starved overnight, and treated with 40 μM forskolin plus 100 μM IBMX for 30 min or 100 ng of TPA per ml for 15 min. Raf-1 immunoprecipitates were sequentially immunoblotted with phospho-Raf-1 S43 and Raf-1 antibodies. (B) COS-1 cells transfected with pCMV5-Raf-1 or pCMV5-RafS43A were serum starved overnight and treated with 100 μM 8Cl-cAMP, 100 ng of TPA per ml, or 20 ng of EGF per ml for 30 min as indicated. For cotreatments 8Cl-cAMP was administered first (20 min). Cell lysates were incubated with Ras/GTP beads for 1 h, and the pulldowns were analyzed by immunoblotting with a Raf-1 antibody. In the lower panel cell lysates were examined for the expression of transfected Raf proteins. (B) An aliquot of the cell lysates was immunoprecipitated with crafVI antibody to assay kinase activity by using a linked assay with MEK and kinase-negative ERK (ERK−) as substrates. The kinase assays were blotted, autoradiographed (upper panel), and stained with crafVI (lower panel). vect., vector-transfected cells; S43A, pCMV5-RafS43A-transfected cells.