SHIP downregulates FcγR-induced IL-1β and IL-6 production through the inhibition of PI3K and Ras/MAPK pathways. SHIP^+/+ and SHIP^–/– macrophages were treated with either Me₂SO (DMSO), 10 μM LY294002, or 2.5 μM UO126 for 30 minutes at 37°C prior to stimulation with heat-aggregated IgG. (A) The levels of IL-1β in cells treated with DMSO or UO126 (i) and IL-1β in cells treated with DMSO or LY294002 (ii) were measured by ELISA. The graphs represent the mean and SEM of values obtained from 3 independent experiments. Data were analyzed by Student t test. *P ≤ .05. (B) The levels of IL-6 in supernatants of cells treated with DMSO or UO126 (i) and cells treated with DMSO or LY294002 (ii) were measured by ELISA. The graphs represent the mean and SEM of values obtained from 3 independent experiments. Data were analyzed by Student t test. *P ≤ .05. (C) Protein-matched lysates were analyzed by Western blotting with phospho-Erk antibody (top panels). Parallel samples were probed with antiphospho serine Akt antibody (middle panels), and the membrane was reprobed with anti-Akt antibody (bottom panels). (D) Raw 264.7 stable transfectants overexpressing vector alone, wild-type SHIP, or catalytic-deficient D675A SHIP were stimulated with heat-aggregated IgG. Production of IL-1β and IL-6 was analyzed by measuring lysates and supernatants, respectively, by ELISA at 5 hours and 8 hours after stimulation. Data were analyzed by Student t test. *P ≤ .05.