Engineered Src::dGBP1-HA is a functional enzyme and efficiently phosphorylates Bsk::GFP in vivo while being innocuous in the absence of the GFP target. (a) Panels show lateral views of fixed Drosophila embryos at stage 13–14 (dorsal closure) expressing synthetic Src kinase variants without the nanobody in the engrailed domain. Embryos were stained with anti-phospho-JNK antibody that recognizes Bsk, the Drosophila JNK ortholog and an anti-phosphotyrosine antibody (pY), that detects tyrosine phosphorylated proteins in all species. (b) Panels show lateral views of fixed Drosophila embryos at stage 13–14 (dorsal closure) expressing Bsk::GFP and synthetic Src kinase variants with C-terminal dGBP1 expressed in the engrailed domain. Embryos were stained as in panel a. The right panel in both a and b shows magnification of the respective pJNK and pY images for each of the embryo genotypes shown on the left. Arrows point to the areas of increased pJNK and pY signal in the engrailed domain where the active kinase is expressed. For every expressed synthetic kinase, the number of considered embryos is indicated (n). Scale bar, 25 μm; in the zoomed panel, 10 μm.