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. 2015 Feb 9;125(3):1215–1227. doi: 10.1172/JCI76693

Figure 1. Intestinal epithelial wounding induces release of ANXA1-bearing EVs.

Figure 1

(A) Immunoblotting for ANXA1 protein in supernatant of nonwounded (nw) or wounded (wound) human epithelial cells. Densitometry analysis shows that ANXA1 protein in cell supernatants derived from wounded cells is significantly increased versus nonwounded confluent cells (2.94-fold increase, P < 0.0001, average of n = 5 immunoblots), and no significant change was observed in the lysate. (B) Immunoblotting for ANXA1 protein in EVs isolated from supernatant. Densitometry analysis shows that ANXA1 protein on EVs derived from wounded cells is increased versus nonwounded cells (2.166-fold increase, P = 0.0066, average of n = 4 immunoblots). (C) Immunogold labeling for ANXA1 and transmission electron microscopy to detect ANXA1 distribution in EVs. Scale bar: 100 nm. (D) Immunoblotting for conventional EV markers in epithelial cell lysates (representative of n = 4 immunoblots). sADAM-10, soluble ADAM-10. (E) EVs were incubated with CD63+ Dynabeads and analyzed by immunoblotting. Densitometry analysis shows that ANXA1 protein on EVs derived from wounded cells is increased versus nonwounded cells (1.459-fold increase, P = 0.0004, average of n = 4 immunoblots). (F) Biotinylated cell surface proteins released from the supernatant of nonwounded and wounded IECs captured with avidin sepharose beads. Densitometry analysis shows that ANXA1 protein on EVs derived from wounded cells is increased versus nonwounded cells (4.239-fold increase increase, P = 0.0002, average of n = 4 immunoblots). (G) Immunoblots show ANXA1 and CD9 proteins in EVs after IEC wounding and treatment with BAPTA-AM (30 μM), Z-VAD-FMK (25 mM), cytochalasin D (2 μM), jasplakinolide (1 μM), dynasore (80 μM), ML-7 (20 μM), and Y-27632 (10 μM). All the results in this figure are representative of at least 3 independent experiments.