Detection of RTK pathway activation in Drosophila using anti-dpERK immunofluorescence staining.
In Drosophila, like in other metazoans, receptor tyrosine kinase (RTK) signaling pathways control diverse cellular processes such as migration, growth, fate determination, and differentiation (Shilo, Development 132:4017-4027, 2005). Activation of RTKs by their extracellular ligands triggers a signal transduction cascade, mediated by the Ras/Raf/MEK cassette, which ultimately leads to dual phosphorylation and activation of the mitogen-activated protein kinase/extracellularly regulated kinase (MAPK/Erk). Once active, MAPK/Erk phosphorylates its cytoplasmic and nuclear substrates, consequently modulating (i.e., stimulating or inhibiting) their biological function (Murphy and Blenis, Trends in Biochemical Sciences 31:268-275, 2006). The currently available antibody specific for the doubly phosphorylated form of MAPK/Erk (dpERK) (Yung et al., FEBS Letters 408:292-296, 1997) provides a valuable readout for RTK signaling: it enables the spatiotemporal detection of RTK pathway activity in the developing organism, in situ (Gabay et al., Development 124:3535-3541, 1997; Gabay et al., Science 277:1103-1106, 1997). Here, we present a detailed protocol for anti-dpERK immunofluorescent staining that can be applied to the analysis of MAPK/Erk signaling in Drosophila embryogenesis.
Helman
Aharon
A
Department of Developmental Biology and Cancer Research, IMRIC, Faculty of Medicine, The Hebrew University, Jerusalem, Israel.
Paroush
Ze'ev
Z
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology* / Q000196:embryology / Q000201:enzymology / Q000378:metabolism; D004625:Embryo, Nonmammalian; D048049:Extracellular Signal-Regulated MAP Kinases / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D010750:Phosphoproteins / Q000276:immunology*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation
D000906:Antibodies; D010750:Phosphoproteins; D020794:Receptor Protein-Tyrosine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases