Calpain-1/2 cleave annexins, which are then shed in MVs. (A) Immunoblot analysis of cytosol fractions after incubation with or without 1 mM Ca²⁺, with a range of concentrations of ALLN inhibitor (Calpain Inh.). (B) Coomassie-stained gel showing purification of annexin A2-Halo from E. coli (In—lysate input; FT—100k × g pellet fraction flow through; W—CaCl₂-containing 100k × g pellet wash; ATP Elu—elution from 100k × g pellet fraction with increasing concentrations of ATP). (C) Coomassie-stained gel showing purification of annexin A6-HA from E. coli (In—lysate input; FT—100k × g pellet flow through; W—CaCl₂-containing 100k × g pellet wash; Elu—elution off 100k × g pellet with 10 mM ATP). (D) Coomassie-stained gel showing the mobility of recombinant annexin A6-HA, incubated with a range of concentrations of purified, porcine calpain-1. Arrows indicate uncleaved and cleaved products. (E) Schematic illustrating substrate binding and elution of substrates (yellow) to catalytic cysteine-to-serine mutant calpain baits (dCAPN, purple). (F) Coomassie-stained gel showing initial his-tag purification of CAPN1[C115S]-3xFlag-6xHis (dCalpain-I) and CAPN2[C105S]-3xFlag-6xHis (dCalpain-II) in complex with CAPNS1(86–268) from E. coli (In—lysate input; FT—bead flow through; W—bead wash; Elu—elution from Ni²⁺ with 300 mM imidazole). Arrows indicate calpain proteins. Source data are available for this figure: SourceData FS4.