DOCK5 acts as an adaptor to mediate phosphorylation and inactivation of GSK3β. (A) After expression of FLAG-tagged Nck2 and/or V5-tagged GSK3β in HEK-293T cells with GFP-tagged WT or mutant DOCK5, cell extracts were immunoprecipitated with anti-FLAG antibody. The precipitated proteins (top two panels) and total lysate control (bottom three panels) were analyzed by immunoblot for GSK3β, Nck2, or DOCK5. (B) 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-GSK3β antibody (top). The recombinant proteins used in this assay were detected with CBB staining (bottom). (C) After expression of GFP-tagged WT or mutant DOCK5 in HEK-293T cells with FLAG-tagged Nck2 and/or V5-tagged GSK3β, cell extracts were immunoprecipitated with anti-GFP antibody. The precipitated proteins (top two panels) and total lysate control (bottom three panels) were analyzed by immunoblot for GSK3β, DOCK5, and Nck2. (A–C) Data are representative of six (A), two (B), or four (C) independent experiments. (D) After expression of GFP-tagged WT DOCK5 in HEK-293T cells with V5-tagged Akt, RSK2, or S6K, cell extracts were immunoprecipitated with anti-GFP antibody. The precipitated proteins (top two panels) and total lysate control (bottom two panels) were analyzed by immunoblot for DOCK5 or V5-tagged proteins. (E) The MC/9 cell lysates were pulled down with GST alone or GST-fusion Akt, 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). (F) After expression of FLAG-tagged Nck2 and/or V5-tagged Akt in HEK-293T cells with GFP-tagged WT or mutant DOCK5, cell extracts were immunoprecipitated with anti-FLAG antibody. The precipitated proteins (top two panels) and total lysate control (bottom three panels) were analyzed by immunoblot for Akt, Nck2, or DOCK5. (D–F) Data are representative of three (D), two (E), or five (F) independent experiments. (G) IgE-sensitized WT and Dock5^−/− BMMCs were stimulated with DNP-HSA for the indicated times, lysed, and analyzed by immunoblot for phosphorylated and total GSK3β. (H) GSK3β phosphorylation in WT BMMCs treated with 100 nM wortmannin was assayed by immunoblot. Total GSK3β is show as a control. (I) IgE-sensitized MC/9 cells stably expressing WT or mutant Nck2 were stimulated with DNP-HSA for the indicated times, lysed, and analyzed by immunoblot for phosphorylated and total GSK3β. (G–I) Data are representative of four (G), three (H), or five (I) independent experiments. (J and K) IgE-sensitized Dock5^−/− BMMCs were treated with various concentrations of LiCl (J) or SB216763 (K) and were then stimulated with DNP-HSA for 1 h before β-hexosaminidase release assays. As a control, WT BMMCs treated with vehicle alone (H₂O for LiCl and DMSO for SB216763) were similarly analyzed. Data (mean ± SD of triplicate samples) are representative of five independent experiments; *, P < 0.05; **, P < 0.01.