Effect of DOCK5 deficiency on FcεRI-mediated proximal signaling. (A) IgE-sensitized WT and Dock5^−/− BMMCs were stimulated with DNP-HSA for the indicated times, lysed, and analyzed by immunoblot for phosphorylated and total Lyn, Syk, and PLC-γ2. Data are representative of two (for Lyn and PLC-γ2) or three (for Syk) independent experiments. (B) IgE-sensitized WT and Dock5^−/− BMMCs were loaded with Fura2-AM, suspended with Tyrode’s buffer, and stimulated with DNP-HSA. Data are indicated as the Fura-2 ratio at 340:380 nm and are representative of five independent experiments. (C) IgE-sensitized WT and Dock5^−/− BMMCs were stimulated with DNP-HSA for the indicated times, lysed, and analyzed by immunoblot for phosphorylated and total Erk, p38, Gab2, and Akt. Data are representative of two (for Erk, p38, and Akt) or three (for Gab2) independent experiments. (D) IgE-sensitized WT and Dock5^−/− BMMCs were labeled with ³²P_i and stimulated with DNP-HSA. Phospholipids were extracted at the indicated times and separated on a thin-layer chromatography plate for PIP₃ measurement. Data are representative of two independent experiments.