Skip to main content
. 2023 May 19;12:e86556. doi: 10.7554/eLife.86556

Figure 4. ANXA6 depletion blocks ionomycin-mediated exosome secretion and stalls multivesicular bodies (MVBs) at the cell periphery.

(A) Immunoblot analysis of ANXA6 and GAPDH expression from GFP, ANXA6-I, and ANXA6-II shRNA CD63-nanoluciferase (Nluc) cells is shown. (B) Immunoblot analysis of CPNE3 and GAPDH expression from GFP, CPNE3-I, and CPNE3-II shRNA CD63-Nluc cells are shown. (C) Normalized exosome production derived from GFP, ANXA6-I, ANXA6-II, CPNE3-I, and CPNE3-II shRNA CD63-Nluc cells grown in conditioned medium for 24 hr are shown. (D) Normalized exosome production derived from GFP, ANXA6-I, ANXA6-II, CPNE3-I, and CPNE3-II shRNA CD63-Nluc cells treated with 5 µM ionomycin for 30 min are shown. Data plotted represent the means from three independent experiments, and error bars represent SDs. Statistical significance was performed using an ANOVA (*p<0.05, ****p<0.0001, and ns = not significant). (E) CD63 immunofluorescence and phalloidin staining of GFP or ANXA6-I shRNA CD63-Nluc cells after 30 min of DMSO or 5 µM ionomycin treatment are shown. White arrows indicate peripheral CD63 puncta. Scale bars: 10 µm.

Figure 4—source data 1. Uncropped immunoblot images corresponding to Figure 4.

Figure 4.

Figure 4—figure supplement 1. Exosome secretion from polyclonal ANXA6 KO cells.

Figure 4—figure supplement 1.

(A) Immunoblot analysis of ANXA6 and vinculin expression from sgNT and sgANXA6 CD63-Nluc cells are shown. (B) Normalized exosome production derived from sgNT and sgANXA6 CD63-Nluc cells treated with 5 µM ionomycin for 30 min is shown. Data plotted represent the means from three independent experiments, and error bars represent SDs. Statistical significance was performed using a Student’s T-test (**p<0.01).
Figure 4—figure supplement 1—source data 1. Uncropped immunoblot images corresponding to Figure 4—figure supplement 1.