Figure 2.

GrM cleaves host cell protein hnRNP K. (a) HeLa cell lysates were incubated with GrM (1 μM) or GrM-SA (1 μM) for 1 h at 37°C and then subjected to 2D-DIGE. A representative protein spot that is identified by mass spectrometry as a cleavage fragment of hnRNP K is depicted. (b) Jurkat lysates (5 μg) were incubated with increasing concentrations of GrM or GrM-SA (300 nM) for 4 h at 37°C and immunoblotted for hnRNP K. (c) Purified His-hnRNP K (1.5 μg) was incubated with increasing concentrations of GrM or GrM-SA (300 nM) for 16 h at 37°C and subjected to SDS-PAGE. Proteins were stained with InstantBlue. (d) Schematic overview of GrM cleavage sites within hnRNP K (NLS, nuclear localization signal; KH, K homology domain; KI, K interactive region; KNS, K nuclear shuttling signal; RGG, arginine–glycine–glycine). (e) HeLa cells were transfected with His-tagged hnRNP K wild-type (wt) or mutant expression plasmids and lysed 2 days post-transfection. Expression of recombinant proteins was determined by subjecting untreated lysates to immunoblotting using anti-6 × His antibody (bottom panel). Lysates were incubated with 300 nM GrM or left untreated for 4 h at 37°C and immunoblotted using anti-6 × His antibody (upper panel) (NS, nonspecific). (f) HFFs were incubated with GrM (2.8 μM) or GrM-SA (2.8 μM) in the presence or absence of SLO (2 μg/ml) for 16 h at 37°C. Lysates were immunoblotted for hnRNP K. (g) HFFs were incubated with GrM (1 μM), GrM-SA (1 μM), GrB (100 nM), or left untreated in the presence or absence of SLO (1 μg/ml) with or without zVAD-fmk (100 μM) for 16 h at 37°C. Cell viability was assessed by staining cells with annexin-V-FLUOS (fluorescein) and PI followed by flow cytometry analysis. Annexin-V and PI double-negative cells were considered to be viable. Bars represent the mean±S.D. of three independent experiments. (h) HFFs were incubated with GrM (1 μM), GrM-SA (1 μM), GrB (100 nM), or left untreated in the presence or absence of SLO (1 μg/ml) with or without zVAD-fmk (100 μM) for 16 h at 37°C. Cells were lysed and immunoblotted using antibodies against active caspase-3 (D175). (i) HFFs were challenged with increasing effector:target (E:T) ratios of LAK cells for 4 h at 37°C. Lysates were subjected to immunoblotting using hnRNP K antibodies. Data depicted are representative of at least two independent experiments