Munc13-4 translocation to membrane is Ca²⁺ dependent. (A) Live-cell epifluorescence imaging of GFP-Munc13-4 in MDA-MB-231 cells at indicated times after ionomycin stimulation. See Video 1. (B) MDA-MB-231 cells expressing wild-type GFP-Munc13-4, GFP-Munc13-4 C2A*, or GFP-Munc13-4 C2B* either left untreated or stimulated with 1.25 µM ionomycin for 5 min were fixed and imaged by confocal microscopy. (C) Indicated proteins were detected by SDS-PAGE and Western blotting of lysates with Munc13-4 antibody from MDA-MB-231 cells stably expressing control shRNA (Ctrl) or shRNA targeting Munc13-4 (KD), or Munc13-4 KD cells rescued with shRNA-resistant wild-type Munc13-4, Munc13-4 C2A*, or Munc13-4 C2B*. (D) Culture media supernatants (as in Fig. 1 B) from MDA-MB-231 cells as in C either untreated or stimulated with 1.25 µM ionomycin for 30 min were filtered onto membrane and analyzed for CD63 or GM130. (E) Quantification of CD63⁺ exosome release (from Fig 1 D) shown as mean values ± SE for n = 3. *, P < 0.05 for comparison between ionomycin-treated and basal. Bars, 5 μm.