American Society of Hematology

Figure 3

$Figure 3. PIP5Kγ controls Munc13-4 membrane raft compartmentalization. (A) YTS cells were infected with lentiviruses encoding shRNA sequences targeting PIP5Kβ (shRNA-ctr), PIP5Kα (shRNA-PIP5Kα), or PIP5Kγ (shRNA-PIP5Kγ). Total cell lysates of infected populations were analyzed by immunoblotting with the indicated Abs. The same membrane was reprobed with anti-Vav mAb as loading control. (B) Uninfected, shRNA-ctr, shRNA-PIP5Kα–infected, and shRNA-PIP5Kγ–infected cells were tested in a degranulation assay. After 4 hours of PMA/ionomycin treatment (gray column), the percentage of CD107a+ cells was evaluated by cytofluorimetric analysis. Unstimulated samples (white column) were treated as described in the “Cell stimulation and immunoprecipitation.” Data represent the percentage (means ± SD) from 3 independent experiments. Differences between uninfected or shRNA-ctr and shRNA-PIP5Kγ-silenced cells were significant (**P < .01; ***P < .005). (C) shRNA-ctr, shRNA-PIP5Kα-, and shRNA-PIP5Kγ–silenced cells were treated with PMA/ionomycin. Unstimulated samples (−) were treated as described in “Methods.” Raft and soluble compartments were isolated and equal amount of proteins were analyzed by immunoblotting with anti–Munc13-4 Ab. One representative experiment of 3 performed is shown. Percentage of raft-associated Munc13-4 in unstimulated (white column) and PMA/ionomycin–treated cells (gray column) was obtained by densitometric analysis (right panel) as described in Figure 2B. The difference (*P = .05) between unstimulated shRNA-ctr and shRNA-PIP5Kγ populations was significant. Data from 3 independent experiments (means ± SD) are shown (right panel). (D) Primary cultured NK cells were tested in a CD16-induced redirected killing assay toward P815 target cells in the presence of the indicated amounts of BAPTA/AM (left panel). NK cells were pretreated with BAPTA/AM (20μM) or control medium (vehicle) and left unstimulated (−) or stimulated with anti-CD16. Distribution of Munc13-4 in raft and soluble fractions was analyzed by immunoblotting (middle and right panels). One representative experiment of 3 performed is shown.$

PIP5Kγ controls Munc13-4 membrane raft compartmentalization. (A) YTS cells were infected with lentiviruses encoding shRNA sequences targeting PIP5Kβ (shRNA-ctr), PIP5Kα (shRNA-PIP5Kα), or PIP5Kγ (shRNA-PIP5Kγ). Total cell lysates of infected populations were analyzed by immunoblotting with the indicated Abs. The same membrane was reprobed with anti-Vav mAb as loading control. (B) Uninfected, shRNA-ctr, shRNA-PIP5Kα–infected, and shRNA-PIP5Kγ–infected cells were tested in a degranulation assay. After 4 hours of PMA/ionomycin treatment (gray column), the percentage of CD107a⁺ cells was evaluated by cytofluorimetric analysis. Unstimulated samples (white column) were treated as described in the “Cell stimulation and immunoprecipitation.” Data represent the percentage (means ± SD) from 3 independent experiments. Differences between uninfected or shRNA-ctr and shRNA-PIP5Kγ-silenced cells were significant (**P < .01; ***P < .005). (C) shRNA-ctr, shRNA-PIP5Kα-, and shRNA-PIP5Kγ–silenced cells were treated with PMA/ionomycin. Unstimulated samples (−) were treated as described in “Methods.” Raft and soluble compartments were isolated and equal amount of proteins were analyzed by immunoblotting with anti–Munc13-4 Ab. One representative experiment of 3 performed is shown. Percentage of raft-associated Munc13-4 in unstimulated (white column) and PMA/ionomycin–treated cells (gray column) was obtained by densitometric analysis (right panel) as described in Figure 2B. The difference (*P = .05) between unstimulated shRNA-ctr and shRNA-PIP5Kγ populations was significant. Data from 3 independent experiments (means ± SD) are shown (right panel). (D) Primary cultured NK cells were tested in a CD16-induced redirected killing assay toward P815 target cells in the presence of the indicated amounts of BAPTA/AM (left panel). NK cells were pretreated with BAPTA/AM (20μM) or control medium (vehicle) and left unstimulated (−) or stimulated with anti-CD16. Distribution of Munc13-4 in raft and soluble fractions was analyzed by immunoblotting (middle and right panels). One representative experiment of 3 performed is shown.

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