American Society of Hematology

Figure 5.

$Figure 5. Serglycin-bound grB is taken up predominantly by a dynamin-dependent mechanism. hCTLs were induced to degranulate over immobilized anti-CD3 mAb, and the supernatant was collected and fractionated over a 100-kDa cut-off centrifugal filter. HeLa-DynWT or DN cells were treated to suppress (+tet) or induce (–tet) overexpression from the transfected dynamin cDNA and then incubated for 3 hours at 37° C with the high-molecular-weight fraction of degranulate material (HighDegran). Caspase activation was quantified by labeling with M30 mAb followed by flow cytometry. (A) Equivalent volumes of hCTL degranulate (Degran), low-molecular-weight degranulate fraction (LowDegran), and HighDegran were resolved on a gel prior to immunoblotting. (Ai) Samples, preincubated ± 2 M sodium chloride, were resolved on a 1% agarose gel in TBE, pH 7.4, and the blot was probed for grB. (Aii) Samples were resolved by SDS-PAGE, and the blot was probed for grB or pfn. (B) Cells were treated with various concentrations of HighDegran measured in nanograms per milliliter equivalents of grB activity. (C) HeLa-DynDN cells were treated with HighDegran (500 ng/mL grB activity) that had been pretreated with DMSO or grB-specific inhibitor (grB-I; 20 μM). (D) HeLa-DynDN +tet cells were treated with purified grB (500 ng/mL) pretreated with DMSO or grB-specific inhibitor (grB-I; 20 μM), along with AdV (500 pfu per cell). The mean ± SD of 5 (panel B, WT), 6 (panel B, DN), or 3 (C-D) independent experiments is shown (*.01 < P < .05; ***P < .001).$

Serglycin-bound grB is taken up predominantly by a dynamin-dependent mechanism. hCTLs were induced to degranulate over immobilized anti-CD3 mAb, and the supernatant was collected and fractionated over a 100-kDa cut-off centrifugal filter. HeLa-DynWT or DN cells were treated to suppress (+tet) or induce (–tet) overexpression from the transfected dynamin cDNA and then incubated for 3 hours at 37° C with the high-molecular-weight fraction of degranulate material (HighDegran). Caspase activation was quantified by labeling with M30 mAb followed by flow cytometry. (A) Equivalent volumes of hCTL degranulate (Degran), low-molecular-weight degranulate fraction (LowDegran), and HighDegran were resolved on a gel prior to immunoblotting. (Ai) Samples, preincubated ± 2 M sodium chloride, were resolved on a 1% agarose gel in TBE, pH 7.4, and the blot was probed for grB. (Aii) Samples were resolved by SDS-PAGE, and the blot was probed for grB or pfn. (B) Cells were treated with various concentrations of HighDegran measured in nanograms per milliliter equivalents of grB activity. (C) HeLa-DynDN cells were treated with HighDegran (500 ng/mL grB activity) that had been pretreated with DMSO or grB-specific inhibitor (grB-I; 20 μM). (D) HeLa-DynDN +tet cells were treated with purified grB (500 ng/mL) pretreated with DMSO or grB-specific inhibitor (grB-I; 20 μM), along with AdV (500 pfu per cell). The mean ± SD of 5 (panel B, WT), 6 (panel B, DN), or 3 (C-D) independent experiments is shown (*.01 < P < .05; ***P < .001).

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