Expression of N-Rok::vhhGFP4^Vi in Tkv::YFP embryos has no effect on pMad levels. (a) Panels show lateral views of fixed Tkv::YFP embryos at stage 13/14 (dorsal closure) expressing N-Rok::vhhGFP4^Vi in the en domain (visualized by co-expression of mCherry-nls). Embryos were stained with anti-phospho-Mad antibody. (b) Panels show lateral views of fixed embryos at stage 13/14 (dorsal closure) expressing Tkv::GFP and N-Rok::vhhGFP4^Vi in the en domain. Embryos were stained with anti-phospho-Sqh/MRLC antibody. (c) Panels show stills from live-imaging with dorsal views of developing DE-Cad::GFP embryos at stages 13/14–16 (dorsal closure) expressing N-Rok::vhhGFP4^Vi in the en domain (visualized by co-expression of mCherry-nls). (d) Panels show lateral views of fixed DE-Cad::GFP embryo at stage 13/14. Embryos were stained with anti-phospho-Sqh/MRLC antibody. Note the enhancement of fluorescent signal due to the binding of vhhGFP4 to DE-Cad::GFP in the en domain. The number of considered embryos is indicated (n). Scale bars, 20 μm in a, b, and d; 50 μm in c.