DOCK5 regulates mast cell degranulation through association with Nck2. (A) IgE-sensitized WT and Dock5^−/− BMMCs were stimulated with DNP-HSA for the indicated times and lysed. Aliquots of the cell extracts were kept for total lysate control, and the remaining extracts were incubated with GST-fusion Rac-binding domain of PAK1. The bound proteins and the total lysate control were probed by immunoblot with anti-Rac1 antibody. Data are representative of five independent experiments. (B) Schematic representation of DOCK5 mutants used in this experiment. (C) Recombinant WT (DHR-2^WT) and mutant (DHR-2^V1559A) DOCK5 DHR-2 proteins were incubated with GDP-loaded GST-fusion Rac1 in the presence of BODIPY-FL–GTP. The change of BODIPY-FL fluorescence was monitored to measure the level of GTP–GDP exchange reaction. Data are representative of two independent experiments. RFU, relative fluorescence unit. (D) The expression of GFP-tagged WT and mutant DOCK5 in Dock5^−/− BMMCs was measured by flow cytometry after adenoviral transfer. As a control, WT BMMCs expressing GFP alone were also analyzed. Data are representative of six independent experiments. VA, V1559A mutant. (E) Release of β-hexosaminidase from Dock5^−/− BMMCs expressing WT or mutant DOCK5 was measured with p-nitrophenyl-N-acetyl-β-d-glucosaminide 1 h after stimulation with anti-DNP IgE plus DNP-HSA. As a control, WT BMMCs expressing GFP alone were also analyzed. Data (mean ± SD of triplicate samples) are representative of six independent experiments; **, P < 0.01. (F) After expression of GFP-tagged DOCK5 in HEK-293T cells with V5-tagged Nck1 or Nck2, cell extracts were immunoprecipitated with anti-GFP antibody. The precipitated proteins (top and middle) and total lysate control (bottom) were analyzed by immunoblot for Nck1, Nck2, or DOCK5. (G) After expression of FLAG-tagged Nck2 in HEK-293T cells with GFP-tagged WT or mutant DOCK5, cell extracts were immunoprecipitated with anti-FLAG antibody. The precipitated proteins (top and middle) and total lysate control (bottom) were analyzed by immunoblot for DOCK5 or Nck2. (H) After expression of GFP-tagged DOCK5 in HEK-293T cells with FLAG-tagged WT or mutant Nck2, cell extracts were immunoprecipitated with anti-FLAG antibody. The precipitated proteins (top and middle) and total lysate control (bottom) were analyzed by immunoblot for DOCK5 or Nck2. (F–H) Data are representative of three (F and H) or eight (G) independent experiments. (I) The MC/9 cell lysates were pulled down with GST alone or GST-fusion Nck2 (WT and W148K mutant), and the bound proteins and total lysate control (input) were probed by immunoblot with anti-DOCK5 antibody (top). The recombinant proteins used in this assay were detected with CBB staining (bottom). Data are representative of two independent experiments. (J) Release of β-hexosaminidase from MC/9 cells stably expressing WT or mutant Nck2 was measured with p-nitrophenyl-N-acetyl-β-d-glucosaminide 1 h after stimulation with anti-DNP IgE plus DNP-HSA. Data (mean ± SD of triplicate samples) are representative of two independent experiments; *, P < 0.05. (K) Organization of microtubules in MC/9 cells stably expressing WT or mutant Nck2 was visualized by microscopy at the indicated time points after stimulation with anti-DNP IgE plus DNP-HSA. Cells were stained for tubulin (red) and nuclei (blue). Data are representative of two independent experiments. Bar, 5 µm. (L) MC/9 cells were treated with siRNAs (Nck2-5 and Nck2-7) or control oligonucleotide, lysed, and analyzed by immunoblot for Nck2 or actin as a control. Data are representative of three independent experiments. (M) Release of β-hexosaminidase from Nck2–knocked down MC/9 cells was measured with p-nitrophenyl-N-acetyl-β-d-glucosaminide 1 h after stimulation with anti-DNP IgE plus DNP-HSA. Data (mean ± SD of triplicate samples) are representative of two independent experiments; **, P < 0.01.