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A-Raf, B-Raf and c-Raf (Raf-1) are the main effectors recruited by GTP-bound Ras to activate the MEKMAP kinase pathway. Activation of c-Raf is the best characterized, and involves phosphorylation at multiple activating sites. Six residues of c-Raf (Ser29, Ser43, Ser289, Ser296, Ser301 and Ser642) become hyperphosphorylated in a manner consistent with c-Raf inactivation and the hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c-Raf unresponsive to subsequent activation events. The c-Raf [pSer289/296/301] Antibody detects endogenous levels of c-Raf protein when phosphorylated at either Ser289, Ser296, or Ser301.