American Society of Hematology

Figure 2

$Figure 2. The munc13-4–rab27a interaction is required for degranulation. (A) CTLs from a control (WT-CTLs) or munc13-4–deficient patient (FHL3-CTLs) were transfected with CFP-munc13-4 wild-type (WT) or CFP-munc13-4 (FQL > AAA) mutant construct. In each experiment, the CFP+ (transfected) and CFP− (nontransfected) cell populations were tested for CD107 expression after stimulation with anti-CD3. Numbers indicate the percentage of degranulating CTLs. The data shown are representative of 3 independent experiments with similar results. (B) CTLs from a control (WT) or munc13-4–deficient patient (FHL3) were transfected with various CFP-munc13-4 constructs (WT or mutants) as indicated and the transfected (gray) and untransfected (black) cell populations were analyzed as in panel A. Values represent means ( ± SD) percentages of the CD107+ CTLs. The results shown are representative of 3 independent experiments with similar results. (C) RBL-2H3 cells with and without ectopically expressed YFP-munc13-4 were transfected with 2 siRNAs targeting different regions in rat munc13-4 by AMAXA nucleofection. Cell lysates were analyzed by Western blot with a polyclonal antibody against munc13-4 and a mAb against actin. (D) RBL-2H3 cells stably expressing indicated wild-type and mutant YFP-munc13-4 constructs were transfected with siRNA #2 or control siRNA as in panel A. Cells were activated as described in “Methods,” and β-hexosaminidase activity was determined colorimetrically. Percentage secreted is calculated as fraction of total amount of β-hexosaminidase activity present in a parallel dish of the same cell line that was not stimulated. We then set the extent of secretion in the cells treated with scrambled siRNA to 100%, and normalized the other results to this value. ***P < .005.$

The munc13-4–rab27a interaction is required for degranulation. (A) CTLs from a control (WT-CTLs) or munc13-4–deficient patient (FHL3-CTLs) were transfected with CFP-munc13-4 wild-type (WT) or CFP-munc13-4 (FQL > AAA) mutant construct. In each experiment, the CFP⁺ (transfected) and CFP⁻ (nontransfected) cell populations were tested for CD107 expression after stimulation with anti-CD3. Numbers indicate the percentage of degranulating CTLs. The data shown are representative of 3 independent experiments with similar results. (B) CTLs from a control (WT) or munc13-4–deficient patient (FHL3) were transfected with various CFP-munc13-4 constructs (WT or mutants) as indicated and the transfected (gray) and untransfected (black) cell populations were analyzed as in panel A. Values represent means ( ± SD) percentages of the CD107⁺ CTLs. The results shown are representative of 3 independent experiments with similar results. (C) RBL-2H3 cells with and without ectopically expressed YFP-munc13-4 were transfected with 2 siRNAs targeting different regions in rat munc13-4 by AMAXA nucleofection. Cell lysates were analyzed by Western blot with a polyclonal antibody against munc13-4 and a mAb against actin. (D) RBL-2H3 cells stably expressing indicated wild-type and mutant YFP-munc13-4 constructs were transfected with siRNA #2 or control siRNA as in panel A. Cells were activated as described in “Methods,” and β-hexosaminidase activity was determined colorimetrically. Percentage secreted is calculated as fraction of total amount of β-hexosaminidase activity present in a parallel dish of the same cell line that was not stimulated. We then set the extent of secretion in the cells treated with scrambled siRNA to 100%, and normalized the other results to this value. ***P < .005.

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