Ft and Ex directly bind in vitro and interact at apicolateral regions in vivo. (A) Ft^ΔECD interacts with Ex^FL and Ex^FERM. S2R⁺ cell expression and IP of FLAG-tagged Ex^FL or Ex^FERM in the presence of Ft^ΔECD, compared to FLAG-bead controls. Ft presents as multiple bands due to proteolytic processing (Feng and Irvine, 2009; Sopko et al., 2009). n/a indicates no Flag-tagged protein added. (B) Ft^ICD directly binds Ex^NT. In vitro transcribed and translated GFP as a control, Ex^NT and Ex^CT were incubated with bacterially expressed and purified GST alone or GST::Ft^ICD and subjected to GST-purification. The expression and presence of proteins was analyzed by immunoblotting with the indicated antibodies. (C) Schematic representation of Ft, Ft^ΔECD and Ft^ICD and Ex protein. Red dashes within the Ft ICD represent the conserved regions A–F. (D) Ft and Ex interact at apical membrane in vivo. Transverse confocal micrographs of third instar imaginal discs expressing ubi-Ex^1-468::GFP subjected to anti-FLAG and anti-GFP PLA. Genetic control expresses only ubi-Ex^1-468::GFP with wild-type Ft, and the Ex::Ft interaction condition expresses ft::FLAG at the endogenous locus and ubi-Ex^1-468::GFP. Ex^1-468::GFP is observed by direct fluorescence of GFP (gray or green in merge), PLA signal (gray or red in merge) marks interaction loci, and Hoechst (cyan in merge) marks nuclei. Sections are orientated apical up. Scale bars are 10 µm. WB, Western blot. Source data are available for this figure: SourceData F2.