SHIP1 is phosphorylated upon LPS stimulation and inhibits LPS-induced inflammatory cytokine production by macrophages. (A) SHIP1 becomes phosphorylated following LPS stimulation in macrophages. RAW264.7 macrophages were treated with 100 ng/mL LPS for the indicated time periods, lysed, then anti-SHIP1 antibody or control isotype IgG were used to immunoprecipitate (IP) SHIP1 from the extracts. Expression of phosphorylated and total SHIP1 was detected using specific antibodies to phosphotyrosine or SHIP1, respectively, by Western blotting.;65IB indicates immunoblot; and p-, phosphorylated. (B) SHIP1 expression is up-regulated after LPS stimulation in macrophages. RAW264.7 macrophages were treated with 100 ng/mL LPS for the indicated time periods. Expression of SHIP1 was detected by Western blotting. (C) SHIP1-specific interfering RNA inhibits SHIP1 expression in macrophages. RAW264.7 macrophages were transfected with pGFP together with SHIP1 interfering RNA plasmid pU6-SHIP1 or control plasmid pU6. After 48 hours, GPF-expressing cells were sorted and SHIP1 expression was detected by Western blot. Densitometrically analyzed and normalized over β-actin signal in the respective lane, the relative expression level of SHIP1 in the control cells was expressed as 1. (D) SHIP1 knockdown increases LPS-induced inflammatory cytokine production by macrophages. RAW264.7 macrophages were transfected with pU6-SHIP1 or control plasmid pU6 and cultured for 48 hours and then stimulated with 100 ng/mL LPS for 8 hours. Concentrations of TNF-α and IL-6 in the supernatants were measured by ELISA. Ctrl indicates control. Data are shown as mean ± SD (n = 3) of one representative experiment; similar results were observed in 3 separate experiments.