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. Author manuscript; available in PMC: 2016 Jan 2.
Published in final edited form as: Nature. 2015 May 11;523(7558):101–105. doi: 10.1038/nature14357

Figure 1. Calcium rapidly stimulates intramembrane proteolysis in Drosophila cells by endogenous DmRho4.

Figure 1

a Diagram comparing the predicted calcium-binding loop residues of DmRho4 to an EF-hand consensus (in red). b Calcium ionophore treatment of Drosophila S2R+ cells induced cleavage of GFP-Spitz, but not its cleavage-site mutant, by endogenous DmRho4. Graph shows expression levels of Drosophila rhomboid genes in S2R+ cells (RNAseq data from modENCODE, modencode.org). c Ionophore-induced Spitz cleavage was detectable within 5 min (red triangle) and linear for 3h. d RNAi knockdown of DmRho4 but not of DmRho1 abrogated calcium-induced cleavage of GFP-Spitz. e Plasmid expression of DmRho4 rescued calcium-induced cleavage of GFP-Spitz in S2R+ cells undergoing RNAi. f Calcium-stimulated Spitz cleavage abolished by DmRho4 RNAi could not be rescued by DmRho1 overexpression. All images are anti-GFP western analyses, with substrate and cleavage bands denoted by black or open triangles, respectively, and non-specific bands marked by × (see Fig. 3d for untransfected cells).