Figure 3.
Figure 3. DOCK5 regulates mast cell degranulation by controlling microtubule dynamics. (A) Release of β-hexosaminidase from IgE-sensitized WT and Dock5−/− BMMCs was measured with p-nitrophenyl-N-acetyl-β-d-glucosaminide 1 h after stimulation with the indicated concentration of DNP-HSA. (B) Release of histamine from IgE-sensitized WT and Dock5−/− BMMCs was measured by ELISA 1 h after stimulation with the indicated concentration of DNP-HSA. (A and B) Data (mean ± SD of triplicate samples) are representative of four independent experiments; *, P < 0.05; **, P < 0.01. (C and D) Production of IL-6 (C) or IL-13 (D) by IgE-sensitized WT and Dock5−/− BMMCs was measured by ELISA 3 h after stimulation with the indicated concentration of DNP-HSA. Data (mean ± SD of triplicate samples) are representative of three (C) or four (D) independent experiments. (E and F) Production of prostaglandin D2 (E) or leukotriene C4 (F) by IgE-sensitized WT and Dock5−/− BMMCs was measured by ELISA 1 h (E) or 15 min (F) after stimulation with DNP-HSA. Data (mean ± SD of triplicate samples) are representative of two (E) or three (F) independent experiments. (G) Secretory granules in WT and Dock5−/− BMMCs were visualized with LysoTracker at the indicated time points after stimulation with anti-DNP IgE plus DNP-HSA. (H) Organization of microtubules in WT and Dock5−/− BMMCs was visualized by microscopy at the indicated time points after stimulation with anti-DNP IgE plus DNP-HSA. Cells were stained for tubulin (green) and nuclei (blue). (G and H) Data are representative of five (G) or four (H) independent experiments. Bars: (G) 10 µm; (H) 5 µm. (I) IgE-sensitized WT and Dock5−/− BMMCs were stimulated with DNP-HSA for the indicated times and then lysed. Polymeric tubulin in the Triton-insoluble fraction was detected with anti–α-tubulin antibody (top), and an SDS-PAGE containing Triton-soluble proteins was stained with CBB (bottom). Data are representative of two independent experiments. (J) Box plot depicts microtubule growth rates in the cell periphery in WT (n = 10) and Dock5−/− (n = 12) BMMCs stimulated with anti-DNP IgE plus DNP-HSA. Growing ends of microtubules were visualized with GFP-tagged c-APC. Data were collected from four separate experiments, and microtubule growth rates were measured in each cell at three to four different points. Each box plot exhibits the median (central line within each box), the 25th and 75th percentile values (box ends), and the 10th and 90th percentile values (error bars). Data are representative of two sets of independent analyses; **, P < 0.01.

DOCK5 regulates mast cell degranulation by controlling microtubule dynamics. (A) Release of β-hexosaminidase from IgE-sensitized WT and Dock5−/− BMMCs was measured with p-nitrophenyl-N-acetyl-β-d-glucosaminide 1 h after stimulation with the indicated concentration of DNP-HSA. (B) Release of histamine from IgE-sensitized WT and Dock5−/− BMMCs was measured by ELISA 1 h after stimulation with the indicated concentration of DNP-HSA. (A and B) Data (mean ± SD of triplicate samples) are representative of four independent experiments; *, P < 0.05; **, P < 0.01. (C and D) Production of IL-6 (C) or IL-13 (D) by IgE-sensitized WT and Dock5−/− BMMCs was measured by ELISA 3 h after stimulation with the indicated concentration of DNP-HSA. Data (mean ± SD of triplicate samples) are representative of three (C) or four (D) independent experiments. (E and F) Production of prostaglandin D2 (E) or leukotriene C4 (F) by IgE-sensitized WT and Dock5−/− BMMCs was measured by ELISA 1 h (E) or 15 min (F) after stimulation with DNP-HSA. Data (mean ± SD of triplicate samples) are representative of two (E) or three (F) independent experiments. (G) Secretory granules in WT and Dock5−/− BMMCs were visualized with LysoTracker at the indicated time points after stimulation with anti-DNP IgE plus DNP-HSA. (H) Organization of microtubules in WT and Dock5−/− BMMCs was visualized by microscopy at the indicated time points after stimulation with anti-DNP IgE plus DNP-HSA. Cells were stained for tubulin (green) and nuclei (blue). (G and H) Data are representative of five (G) or four (H) independent experiments. Bars: (G) 10 µm; (H) 5 µm. (I) IgE-sensitized WT and Dock5−/− BMMCs were stimulated with DNP-HSA for the indicated times and then lysed. Polymeric tubulin in the Triton-insoluble fraction was detected with anti–α-tubulin antibody (top), and an SDS-PAGE containing Triton-soluble proteins was stained with CBB (bottom). Data are representative of two independent experiments. (J) Box plot depicts microtubule growth rates in the cell periphery in WT (n = 10) and Dock5−/− (n = 12) BMMCs stimulated with anti-DNP IgE plus DNP-HSA. Growing ends of microtubules were visualized with GFP-tagged c-APC. Data were collected from four separate experiments, and microtubule growth rates were measured in each cell at three to four different points. Each box plot exhibits the median (central line within each box), the 25th and 75th percentile values (box ends), and the 10th and 90th percentile values (error bars). Data are representative of two sets of independent analyses; **, P < 0.01.

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