The detection of MAPK signaling.

Mitogen-activated protein kinase (MAPK) cascades are central pathways that participate in the intracellular transmission of extracellular signals. Each of the MAPK signaling cascades seems to consist of three to five tiers of protein kinases that sequentially activate each other by phosphorylation. Since the majority of MAPK cascade components are kinases, the methods used to detect their activation involve determining phosphorylation state and protein kinase activities. The primary method describes the use of immunoblotting with specific anti-phospho antibody to detect activation of MAPK components. Alternative methods described are immunoprecipitation of desired protein kinases followed by phosphorylation of specific substrates and the use of an in-gel kinase assay. These methods have proven useful in the study of the MAPK signaling cascades.