SHIP1 inhibits LPS-induced TNF-α production primarily via a phosphatase activity- and PI-3K-independent mechanism in macrophages. (A) Overexpression of wild-type SHIP1 and phosphatase activity-disrupted mutant SHIP1 inhibits LPS-induced TNF-α production in macrophages. RAW264.7 macrophages (2 × 105) stably transfected with control vector, SHIP1 plasmid, or mutant SHIP1 were stimulated with 100 ng/mL LPS for 8 hours or left unstimulated; concentrations of TNF-α in the supernatants were measured by ELISA. (B) SHIP1 inhibits LPS-induced TNF-α production in the presence of PI-3K inhibitors in macrophages. RAW264.7 macrophages (2 × 105) stably transfected with control vector or SHIP1 plasmid were stimulated with 100 ng/mL LPS in the presence of DMSO, 100 ng/mL wortmannin, 30 μM LY303511, or 30 μM LY294002 for 8 hours, then concentrations of TNF-α in the supernatants were measured by ELISA. (C) LY294002 and wortmannin sufficiently inhibit PI-3K activity in macrophages. RAW264.7 macrophages were pretreated with 30 μM LY294002 (also included in the enzyme reaction), 100 nM wortmannin for 40 minutes, or left untreated. Then the cells were stimulated with 1 μg/mL LPS for 10 minutes. PI-3K activity in the cells was assayed as described in “Materials and methods,” under “Assay of PI-3K activity.” For LY294002-treated cells, LY294002 was also added to the reaction mixture. The amount of PI(3,4,5)P3 produced in the reactions was proportional to PI-3K activity. Data are shown as mean ± SD (n = 3) of one typical experiment. Similar results were observed in 3 separate experiments.
