AQX-016A inhibits immune cell activation. (A) SHIP1^+/+ (▩) and SHIP1^−/− (■) macrophages were pretreated with AQX-016A or carrier 30 minutes prior to stimulation with 10 ng/mL of LPS at 37°C for 2 hours and TNFα production determination by ELISA. Absolute TNFα levels for SHIP1^+/+ and SHIP1^−/− cells were 623 ± 30 and 812 ± 20 pg/mL, respectively. Data are expressed as means (± SEM) and are representative of 3 independent experiments. (B) SHIP1^+/+ and SHIP1^−/− mast cells were preloaded with IgE and Fura-2 as described in “Materials and methods, Stimulation of mast cells by FcϵRI crosslinking” and treated for 30 minutes with 15 μM AQX-016A or carrier. Cells were then stimulated (as indicated by ↓) with 0 ng/mL (- - -) or 10 ng/mL (––) DNP-HSA, and intracellular calcium levels were monitored over time by spectrofluorometry.²³  (C) SHIP1^+/+ and SHIP1^−/− macrophages were pretreated for 30 minutes with AQX-016A or carrier prior to stimulation with 10 ng/mL of LPS for 15 minutes at 37°C. Total-cell lysates were analyzed for the indicated phospho-proteins or proteins by immunoblot analysis. (D) Anti–DNP-IgE loaded SHIP1^+/+ and SHIP1^−/− mast cells were treated for 30 minutes with 30 μM AQX-016A or carrier prior to stimulation with 20 ng/mL DNP-HSA for 5 minutes at 37°C; cell lysates were analyzed as in panel C.