SHIP1 negatively regulates LPS-induced activations of the PI-3K pathway in a phosphatase activity-dependent manner in macrophages. (A) Wild-type SHIP1 and mutant SHIP1 were overexpressed in stably transfected RAW264.7 cells. RAW264.7 macrophages were stably transfected with empty pBK-CMV control plasmid, wild-type SHIP1, or phosphatase-inactive SHIP1 plasmid. Expressions of SHIP1 in the cells were detected with SHIP1 antibody. (B) Overexpression of wild-type SHIP1 and phosphatase-inactive SHIP1 differently affect Akt activity in macrophages. RAW264.7 macrophages were stably transfected with empty pBK-CMV control plasmid, wild-type SHIP1, or phosphatase-inactive SHIP1 plasmid. The cells were stimulated for 10 minutes with 100 ng/mL LPS, 100 ng/mL M-GSF, or left unstimulated and then cell extracts were prepared. Akt activity was assayed as described in “Materials and methods,” under “Assay of Akt kinase activity.” Similar results were observed in 3 separate experiments.