Ft and Ex directly bind at the apical membrane. (A) Ft^ICD directly binds Ex^1-468. In vitro transcribed and translated Ex^1-468 was incubated with bacterially expressed and purified GST alone or GST::Ft^ICD and subjected to GST-purification. (B–B′′) High-resolution colocalization of Ex^1-468 and Ft. XY confocal micrograph of third instar wing imaginal discs stained with Ft (mauve in B′′) with direct fluorescence of Ex^1-468::GFP (green in B′′). (C) Ft and Ex interact at apical membrane in vivo. XY confocal micrographs of third instar imaginal discs expressing ubi-Ex^1-468::GFP subjected to anti-FLAG and anti-GFP PLA. 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, which overlap with Ex signal. Images were denoised, scale bar is 2 µm. (D) Ft^ICD does not directly binds Dlish. In vitro transcribed and translated Ex^1-468 (as a positive control) and Dlish 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. WB, Western blot. Source data are available for this figure: SourceData FS2.