Drosophila 14-3-3 proteins interact with Dlg in the wing disc epithelium. (A) Coimmunoprecipitation of endogenous 14-3-3 proteins and Dlg from wild-type wing disc samples. Western blots were probed with anti–14-3-3 antibody (upper lanes) and anti-Dlg antibody (lower lanes) in input and immunoprecipitates pulled down by anti-Dlg antibody. IP, immunoprecipitation. (B–G) PLA indicates close proximity or interactions of two proteins. PLA between Scrib and Dlg in control (nub-Gal4/+; B) and dlg-knockdown (nub-Gal4>dlg-RNAi; C) wing discs. (D) Quantification of the number of PLA (Scrib/Dlg) spots for control (n = 6) and dlg-RNAi (n = 5) wing discs. PLA between 14-3-3 and Dlg in control (E) and dlg-knockdown (F) wing discs. (G) Quantification of the number of PLA (14-3-3/Dlg) spots for control (n = 5) and dlg-RNAi (n = 5) wing discs. Error bars are SD. **, P < 0.01 by Kolmogorov–Smirnov test. Note that PLA signals in MZ of the disc proper diminished in dlg-RNAi wing discs, but not in PE where nub-Gal4 is not expressed. PE, peripodial epithelium; MZ, mitotic zone. (H and I) Subcellular localization of 14-3-3 proteins detected by anti–14-3-3 antibody staining. 14-3-3 proteins localize to the apical cortex and nucleus during interphase (H) and cytosol, including spindle microtubules (α-tubulin), during mitosis (I). Yellow arrows indicate metaphase cells. Scale bars: 5 µm.