Figure 4. Calpain-1/2 cleaves annexins,... | Rockefeller University Press

Figure 4.

$Calpain-1/2 cleaves annexins, which are then shed in MVs. (A) Immunoblot analysis of cytosol fractions after incubation with or without 1 mM Ca2+. (B) Immunoblot analysis of cytosol fractions after incubation with or without 1 mM Ca2+, with a range of concentrations of calpastatin domain I inhibitor (Calpain Inh.). (C) In gel fluorescence of JF646-labeled, recombinant annexin A2-Halo incubated with indicated concentration of purified, porcine calpain-1. Arrows indicate uncleaved and cleaved products. (D) Mapping of tryptic peptides to the first 50 amino acids of annexin A2. Mass spectrometry analysis of recombinant annexin A2 (red text) is compared with tryptic digest-mass spectrometry analysis of recombinant annexin A2 treated with purified, porcine calpain-1 (yellow highlight). (E) Immunoblot analysis of cell lysate and conditioned medium 100k × g pellet fraction after treating cells with 200 ng/ml SLO in the presence or absence of 10 μM calpain inhibitor, ALLN. (F) Schematic illustrating the recombinant annexin A2-Halo reporter, labeled with a 5WS maleimide quencher on the N terminus and a JF646 halo ligand on the C terminus. (G) JF646 fluorescence of an in vitro reaction containing 5 μM self-quenched annexin A2 with or without 0.5 μM porcine calpain-1. (H) Representative confocal micrographs of dequenched annexin A2-Halo-JF646 fluorescence in cells. Times are relative to the first image taken after addition of 1 mM Ca2+. White arrows in pane II indicate puncta on the cell periphery. Arrow in pane III indicates an EV. Scale bars: 10 μm. (I) Total protein (Sypro Ruby staining) and immunoblot analysis of the substrate-trapping experiment, using 3xFlag (−), 3x-Flag C115S calpain-1 (dCAPN-I), or 3x-Flag C105S calpain-2 (dCAPN-II) as bait, is shown. Arrows indicate calpain proteins. (J) Table listing proteins identified in EGTA elutions from 3xFlag, 3x-Flag C115S calpain-1 (dCAPN-I), or 3x-Flag C105S calpain-2 (dCAPN-II) capture experiments. Proteins are listed by exponentially modified protein abundance index (emPAI) in the dCAPN-I elution. Keratin proteins were not included in the list. Source data are available for this figure: SourceData F4.$

Calpain-1/2 cleaves annexins, which are then shed in MVs. (A) Immunoblot analysis of cytosol fractions after incubation with or without 1 mM Ca²⁺. (B) Immunoblot analysis of cytosol fractions after incubation with or without 1 mM Ca²⁺, with a range of concentrations of calpastatin domain I inhibitor (Calpain Inh.). (C) In gel fluorescence of JF646-labeled, recombinant annexin A2-Halo incubated with indicated concentration of purified, porcine calpain-1. Arrows indicate uncleaved and cleaved products. (D) Mapping of tryptic peptides to the first 50 amino acids of annexin A2. Mass spectrometry analysis of recombinant annexin A2 (red text) is compared with tryptic digest-mass spectrometry analysis of recombinant annexin A2 treated with purified, porcine calpain-1 (yellow highlight). (E) Immunoblot analysis of cell lysate and conditioned medium 100k × g pellet fraction after treating cells with 200 ng/ml SLO in the presence or absence of 10 μM calpain inhibitor, ALLN. (F) Schematic illustrating the recombinant annexin A2-Halo reporter, labeled with a 5WS maleimide quencher on the N terminus and a JF646 halo ligand on the C terminus. (G) JF646 fluorescence of an in vitro reaction containing 5 μM self-quenched annexin A2 with or without 0.5 μM porcine calpain-1. (H) Representative confocal micrographs of dequenched annexin A2-Halo-JF646 fluorescence in cells. Times are relative to the first image taken after addition of 1 mM Ca²⁺. White arrows in pane II indicate puncta on the cell periphery. Arrow in pane III indicates an EV. Scale bars: 10 μm. (I) Total protein (Sypro Ruby staining) and immunoblot analysis of the substrate-trapping experiment, using 3xFlag (−), 3x-Flag C115S calpain-1 (dCAPN-I), or 3x-Flag C105S calpain-2 (dCAPN-II) as bait, is shown. Arrows indicate calpain proteins. (J) Table listing proteins identified in EGTA elutions from 3xFlag, 3x-Flag C115S calpain-1 (dCAPN-I), or 3x-Flag C105S calpain-2 (dCAPN-II) capture experiments. Proteins are listed by exponentially modified protein abundance index (emPAI) in the dCAPN-I elution. Keratin proteins were not included in the list. Source data are available for this figure: SourceData F4.

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