Figure 3 - Krz, Dx and Notch colocalize and form a trimeric complex.

(a–i) Confocal images of Drosophila S2 cells expressing HA–Krz alone (a), Flag–Dx alone (b), Notch (N) alone (c), HA–Krz and Flag–Dx together (d–f), and HA–Krz and Notch in the presence of Flag–Dx (g–i). Images in f and i are merges of those in d and e, and g and h, respectively. Co-expression of Krz and Dx leads to the redistribution of Krz and Dx into intracellular vesicles, where they colocalize (d–f). Notch is also present in those vesicles when co-expressed with HA–Krz and Flag–Dx (g–i). (j–l) Co-localization of Flag–Dx and HA–Krz in wing imaginal discs. UAS–Flag–Dx and UAS–HA–Krz were expressed under the control of the C96–GAL4 driver. Image l is a merge of those in j and k. (m) Krz, Dx and Notch form a trimeric complex. S2N cells were transfected with HA–Krz alone, Flag–Dx alone, or HA–Krz and Flag–Dx together, and were incubated for 45 min with an equal number of either S2 cells (lanes 1–3) or S2Dl cells expressing full-length Delta (lanes 4–6). Cell extracts were immunoprecipitated (IP) with anti-HA antibody and immunoblotted with anti-Notch antibody. Notch was immunoprecipitated with HA–Krz, but only in the presence of Dx (lanes 3 and 6), and the formation of this trimeric complex was not affected by the presence of the ligand (Delta). Note that the Notch fragment with a relative molecular mass of 120,000 (N 120 K) was more efficiently bound than the full-length Notch receptor (N^FL). The bottom panel shows that Notch was present at equal levels in all lysates. Scale bars, 5 m (a–i), 10 m (j–l).