Helman Aharon, Paroush Ze'ev
Department of Developmental Biology and Cancer Research, IMRIC, Faculty of Medicine, The Hebrew University, Jerusalem, Israel.
Methods Mol Biol. 2010;661:401-8. doi: 10.1007/978-1-60761-795-2_24.
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.
在果蝇中，与其他后生动物一样，受体酪氨酸激酶（RTK）信号通路控制着多种细胞过程，如迁移、生长、命运决定和分化（希洛，《发育》132:4017 - 4027，2005年）。RTK被其细胞外配体激活后会触发一个信号转导级联反应，该反应由Ras/Raf/MEK模块介导，最终导致丝裂原活化蛋白激酶/细胞外调节激酶（MAPK/Erk）的双重磷酸化和激活。一旦激活，MAPK/Erk就会磷酸化其细胞质和细胞核底物，从而调节（即刺激或抑制）它们的生物学功能（墨菲和布莱尼斯，《生物化学科学趋势》31:268 - 275，2006年）。目前可获得的针对MAPK/Erk双磷酸化形式（dpERK）的特异性抗体（容等人，《欧洲生物化学学会联合会快报》408:292 - 296，1997年）为RTK信号传导提供了一个有价值的检测指标：它能够在发育中的生物体中原位进行RTK通路活性的时空检测（加贝等人，《发育》124:3535 - 3541，1997年；加贝等人，《科学》277:1103 - 1106，1997年）。在此，我们提供了一份详细的抗dpERK免疫荧光染色方案，可应用于果蝇胚胎发育中MAPK/Erk信号传导的分析。
