Munc13-4 recruitment to recycling endosomes is dependent on Rab11a. (A) MDA-MB-231 cells immunolabeled for endogenous CD63 (red) and Munc13-4 (green) by confocal microscopy. (B) MDA-MB-231 cells expressing GFP-Munc13-4 and mCherry-Rab27a, mCherry-Rab27b, or mApple-Rab11a imaged by confocal microscopy. (C) Percentage of cells with Pearson’s correlation coefficient for GFP versus mCherry/mApple >0.7. (D) MDA-MB-231 cells stably expressing control shRNA (Ctrl) or Rab11a shRNA and GFP-Munc13-4 were left untreated or were stimulated with 1.25 µM ionomycin for 5 min, fixed, and imaged by confocal microscopy. (E) SDS-PAGE Western blot of indicated proteins in cells stably expressing control shRNA (Ctrl) or Rab11a shRNA. (F) Culture media supernatants (as in Fig. 1 B) from control (Ctrl) or MDA-MB-231 cells stimulated with 1.25 µM ionomycin for 30 min were filtered onto membrane and immunoblotted for CD63 and GM130. (G) Quantification of CD63⁺ exosome release as percentage of cellular total indicated as mean values ± SE for n = 5. **, P < 0.01 for comparison of ionomycin-treated samples. Bars, 5 μm.