Fig. 5.
Fig. 5. GzmC does not cleave or activate GzmB substrates. / (A) GzmB-induced caspase-3 activity in target cells. (top panel) YAC1 cells were pretreated with a fixed dose (100 μM) of the broad-spectrum caspase inhibitor D-fmk or the DMSO vehicle for 30 minutes at 37°C. Next, the cells were treated with perforin plus 1 μM rGzmB and incubated at 37°C for 5, 15, 30, or 60 minutes. Cellular lysates were prepared, and DEVD-AMC cleavage was measured in triplicate using a spectrofluorometer. (bottom panel) Before target cell harvest at every time point, a fraction of each sample was stained with 7-AAD and quantified using flow cytometry to assess viability. (B) Absence of GzmC-induced caspase-3 activity. (top panel) As described in panel A, D-fmk– or DMSO-pretreated YAC1 cells were treated with perforin plus 1 μM rGzmC followed by fluorometric analysis. (bottom panel) Portions of the rGzmC-loaded samples were stained with 7-AAD and quantified with flow cytometry. (C) Full-length recombinant p22 BID was incubated at 37°C for 30 minutes alone or with increasing concentrations of active rGzmB or rGzmC. Samples were harvested and electrophoresed on reducing SDS-PAGE gels. Western blot analysis was performed with a rabbit polyclonal anti-BID antibody. Only rGzmB directly processes p22 BID to the truncated BID (tBID) form. (D) rhICAD was processed as above in panel A and immunoblotted using a rabbit polyclonal anti-ICAD antibody. Only rGzmB processes ICAD to its p30 form.

GzmC does not cleave or activate GzmB substrates.

(A) GzmB-induced caspase-3 activity in target cells. (top panel) YAC1 cells were pretreated with a fixed dose (100 μM) of the broad-spectrum caspase inhibitor D-fmk or the DMSO vehicle for 30 minutes at 37°C. Next, the cells were treated with perforin plus 1 μM rGzmB and incubated at 37°C for 5, 15, 30, or 60 minutes. Cellular lysates were prepared, and DEVD-AMC cleavage was measured in triplicate using a spectrofluorometer. (bottom panel) Before target cell harvest at every time point, a fraction of each sample was stained with 7-AAD and quantified using flow cytometry to assess viability. (B) Absence of GzmC-induced caspase-3 activity. (top panel) As described in panel A, D-fmk– or DMSO-pretreated YAC1 cells were treated with perforin plus 1 μM rGzmC followed by fluorometric analysis. (bottom panel) Portions of the rGzmC-loaded samples were stained with 7-AAD and quantified with flow cytometry. (C) Full-length recombinant p22 BID was incubated at 37°C for 30 minutes alone or with increasing concentrations of active rGzmB or rGzmC. Samples were harvested and electrophoresed on reducing SDS-PAGE gels. Western blot analysis was performed with a rabbit polyclonal anti-BID antibody. Only rGzmB directly processes p22 BID to the truncated BID (tBID) form. (D) rhICAD was processed as above in panel A and immunoblotted using a rabbit polyclonal anti-ICAD antibody. Only rGzmB processes ICAD to its p30 form.

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