dEHBP1 accumulates with Delta in the absence of Sec15 and it physically interacts with Sec15 and Rab11. (a–b′′′) xz projection of single ESO clusters within thoracic epithelia, which are wild-type (a–a′′) or homozygous mutant for sec15 (b–b′′, positively marked by the expression of GFP), reveals that dEHBP1 accumulates basally along with Delta in the absence of sec15. (c–c′′) XZ projection of thoracic epithelia, shown in a–b, which contain homozygous mutant cells for sec15 (c′′, positively marked by the expression of GFP), reveals that dEHBP1 accumulates basally in all epithelial cells in pupal thoraces in the absence of sec15. (d–d′′) Sec15-GFP, overexpressed by neur^Gal4, and dEHBP1 colocalize in ESO clusters at the interface of the progeny, as indicated by the arrows. Single-channel representations for dEHBP1 (d′) and Sec15-GFP (d′′) are shown in black and white. (e–e′′) Sec15-GFP and mCherry-dEHBP1 overexpressed by neur^Gal4 colocalize in ESO clusters. (f) HA-Sec15 and FLAG-dEHBP1 coimmunoprecipitate from whole-cell lysates of transiently transfected S2 cells. (g) GST-Rab11 does not interact with in vitro–translated dEHBP1. 10% input of the proteins used for the GST pull-down were analyzed by Coomassie stain to ensure the integrity and equal amounts of GST proteins. (h) dEHBP1 interacts weakly with wild-type and constitutively active Rab11 variants, but strongly with dominant-negative Rab11 in coIP experiments from S2 cells. Bars, 10 µm.