Calpain cleavage attenuates the membrane binding and scabbing activity of annexin A2. (A) Immunoblot analysis of lysates from cells expressing WT ANXA2-HA or ANXA2[P20D, P21D]-HA. Ca²⁺ (1 mM) was added to lysis buffer where indicated. (B) Immunoblot analysis of lysates from cells expressing WT ANXA2-HA or ANXA2-HA with the indicated N-terminal truncations. Ca²⁺ (1 mM) was added to lysis buffer where indicated. (C) Representative confocal micrographs of ANXA2-mScarlet (wt-mScarlet) and ANXA2(18–339)-mNeonGreen (Tr-mNeonGreen)–expressing cells. Image times are relative to the addition of 400 ng/ml SLO. Scale bars: 5 μm. (D) Immunoblot analysis of lysates and anti-HA immunoprecipitation elutions from cells expressing WT ANXA2-HA (wt-HA), ANXA2(18-339)-HA (Tr-HA), ANXA2[P20D, P21D]-HA (Mt-HA), or no HA construct. (E) Representative confocal micrographs of ANXA2-mScarlet (wt-mScarlet) and S100A10-mNeonGreen–expressing cells. Image times are relative to the addition of 400 ng/ml SLO. Arrows indicate cells with annexin A2 and S100A10 translocating to the membrane. Scale bars: 5 μm. (F) Representative widefield micrographs of WT or annexin A2 KO cells, with 2.5 μM Sytox Green added for 6 min. SLO (200 ng/ml) was added with Sytox Green in the indicated panels. Scale bars: 100 μm. (G) Representative confocal micrographs of FM1-43–stained (2.5 μM) WT or annexin A2 KO cells, 100 s after laser ablation. White arrows indicate the ablation site. Scale bars: 5 μm. (H) Quantification over time of the repair scab intensity from FM1-43–stained (2.5 μM) WT or annexin A2 KO cells. Error bars indicate six experimental replicates. (I and J) Representative widefield micrographs (I) and aggregate size quantification (J) of Texas Red-labeled 200-nm liposomes mixed with the indicated combinations of 300-nM annexin A2 (A2), annexin A2-S100A10 (A2+S1), or Calpain-1 (C1). Scale bars: 50 μm. Error bars indicate two experimental replicates. Source data are available for this figure: SourceData F5.