Calpain cleavage attenuates the membrane binding and scabbing activity of annexin A2. (A) Immunoblot analysis of cytosol and membrane fractions, with or without 1 mM Ca²⁺ added prior to fractionation. (B) Representative confocal micrographs of ANXA2-mScarlet (wt-mScarlet) and ANXA2[P20D, P21D]-mNeonGreen (Mt-mScarlet)–expressing cells. Image times are relative to the addition of 400 ng/ml SLO. Scale bars: 5 μm. (C) Total protein (Sypro Ruby staining) analysis of HaloTag “pulldown” on chloroalkane-coated beads is shown, using no bait (−), porcine calpain-1–treated annexin A2-HaloTag bait (treat), or untreated annexin A2-HaloTag bait (untreat). Proteins are labeled with spectral counts, in parentheses, from gel excision-mass spectrometry. (D) Representative widefield micrographs of purified S100A10-mScarlet (1 μM) binding to GUVs with or without annexin A2-HaloTag-JF646 (1 μM). Scale bars: 50 μm. (E) Representative widefield micrographs of WT or annexin A2 KO cells treated with 200 ng/ml SLO and 2.5 μM Sytox Green without Ca²⁺ in the media. Scale bars: 100 μm. (F) Coomassie-stained gels showing purification of annexin A2 or annexin A2–S100A10 complex from E. coli. Panel I shows the initial purification by eluting annexin A2 from the E. coli lysate pellet fraction in a Ca²⁺-dependent manner (Input—lysate input; FT—100k × g pellet flow through; Wash—CaCl₂-containing 100k × g pellet wash; Elution—10 mM ATP elution from 100k × g pellet fraction). Where indicated lysate from S100A10-expressing E. coli was mixed 1:1 with annexin A2 E. coli lysate before purification. Panels II and III show size exclusion chromatography fractions of annexin A2 and annexin A2–S100A10 complex, respectively. (G) Immunoblot analysis shows mechanically lysed cells sequentially fractionated into PNS (diluted 1:5 before loading), 100k × g centrifuged PNS supernatant (100k Sup, diluted 1:5 before loading), and 100k × g pellet washed with 5 mM EGTA and recentrifuged (Pellet Wash). Samples where 1 mM Ca²⁺ was initially added to PNS are indicated. Arrows indicate uncleaved and autolyzed CAPN1. Source data are available for this figure: SourceData FS5.