Fig. 1. Phosphorylation of Groucho in the ventral neuroectoderm depends on EGFR signalling. (A-I) Ventral views of stage 10 embryos; anterior is towards the left. (A-C) Wild-type embryo stained with both pGro (A; red) and dpERK (B; green) antibodies. (C) Merge. There is a significant overlap between the staining in cells that border the midline, whereas only pGro, but not dpERK, is detected in more lateral cells. (D-F) Wild-type embryo double-stained with pGro (D; red) and Gro (E; green) antibodies. (F) Merge. The staining is largely mutually exclusive, indicating that Gro is phosphorylated in cells straddling the ventral midline, whereas in more lateral ectodermal regions it is mostly in its unphosphorylated state. (G-I) Homozygous Egfr^f2 mutant embryos stained for pGro (G; red) and Gro (H; green). (I) Merge. pGro staining is decreased in the ventral neuroectoderm (compare with A,D), and is replaced by Gro staining (compare with E). (F,I) There is complementarity between the pGro and Gro staining, attesting to the specificity of our pGro antibodies (see Fig. 2D below). (C,F,I) Arrowheads indicate the ventral midline. (J) pGro and Gro antibodies differentially recognise the phosphorylated and nonphosphorylated forms of Gro, respectively, in western blot analysis, using a denaturing gel. Bacterially expressed GST-Gro fusion protein is recognised mainly by Gro antibodies (lane 1). Phosphorylation of Gro by ERK2 in vitro leads to its detection primarily by pGro, and prevents its recognition by Gro, antibodies (lane 2). Incubation of phosphorylated Gro with a nonspecific phosphatase (2.5u CIP, lane 3; 5u CIP, lane 4) reverses the recognition by the antibodies, suggesting that phosphorylation itself is enough to cause the differential recognition by the antibodies.