SHIP downregulates FcγR-induced superoxide production. (A) BMMs derived from SHIP^+/+ and SHIP^–/– mice were activated with heat-aggregated IgG for the time points indicated in the figure. Generation of superoxide was measured using 10 μM fluorescent probe DHE. DHE fluorescence intensity is plotted in the graph. (B) BMMs derived from SHIP^+/+ and SHIP^–/– mice were activated with heat-aggregated IgG for 2 hours. Generation of superoxide was measured using fluorescent probe DHE. Data represent mean and SEM of 4 independent experiments. Data were analyzed by Student t test. *P < .05. (C) Protein-matched whole-cell lysates (WCLs) were analyzed by Western blotting with SHIP antibody (i). The same membrane was reprobed with actin antibody (ii). IB indicates immunoblot. (D) Mac-1 expression on the SHIP^+/+ and SHIP^–/– BMMs was analyzed by flow cytometry. For this, the cells were labeled with APC-labeled Mac-1 antibody in the presence of the anti-FcγRII/III monoclonal antibody (mAb) 2.4G2 (to block Fcγ receptors; - - -). Cells were also labeled with APC-labeled isotype control antibody (—).