title: Human and mouse granzyme M display divergent and species-specific substrate specificities. Biochemical Journal, 2011, 437 (3), pp.431-442. &#x27E8;10.1042/BJ20110210&#x27E9;. &#x27E8;hal-00608385&#x27E9;
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  THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20110210 Biochemical Journal Immediate Publication. 2011 Portland Limited us cr ipt against NPM or -tubulin (loading control). Results are representative of three independent experiments. (D) Recombinant human and mouse NPM were incubated for 2 h at 37°C with the indicated concentrations of hGrM or mGrM. Reactions were separated on SDS-PAGE and visualized by Simply Blue staining. Results are representative of three independent experiments. (E) Alignment of human and mouse NPM. Residues boxed in grey are the shared amino acids between human and mouse NPM. Boxed in dark grey is the known hGrM cleavage site [20]. (F) Alignment of the GrM cleavage site in human and mouse NPM. The P1-P4 are completely conserved between mouse and human, but the P1’ and P2’ residues differ. Different mutants of mouse NPM generated are depicted. (G) Purified recombinant wild type (wt) human NPM (hNPM), wt mouse NPM (mNPM) and different NPM mutants were incubated for 2 h at 37°C with the indicated concentrations of human (h) or mouse (m) GrM. Products were separated by SDS-PAGE and visualized by Simply Blue staining. Results are representative of four independent experiments. ce pt ed M an FIG. 7. Human and mouse GrM trigger cell death in human HeLa but not mouse C2C12 tumor cells. Human HeLa cells (A) or mouse C2C12 cells (B) were treated with sublytic concentrations of SLO and various concentrations of hGrM, mGrM or their catalytically inactive mutants (GrM-SA), for 30 min. Cells were treated with hGrB as a positive control. After washing, cells were incubated for another 20 h at 37°C. Cell viability was determined using flow cytometry, with AnnexinV and PI negative cells considered viable.  