Co-culture Activation of MAP Kinase in Drosophila S2 Cells
Intercellular communication often involves phosphorylation of signal transduction proteins, including mitogen-activated protein kinases (MAPKs). Immunological detection of phosphorylated MAPK can be used to monitor signaling in vivo, identify novel pathway components, and assess ligand activity. In this chapter, I describe a cell co-culture method to assess activity of cell-bound extracellular ligands that result in phosphorylation of the ERK (extracellular signal-regulated kinase) MAPK in Drosophila. This protocol may be adaptable to other pathways and/or model systems.
Key wordsMAP kinase ERK dp-ERK S2 cells Co-culture Western blot
I thank Jean-Yves Roignant and Kevin Legent for help developing the ERK activation assay; Benny Shilo for the D2F cells; Jessica Treisman for support during the development of the protocol and critical comments on the manuscript. This work was supported by the March of Dimes Birth Defects Foundation (to Jessica Treisman) and the National Institutes of Health (grant number EY13777 to Jessica Treisman from the National Eye Institute).
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