The WT, but not the D675G, T1, or T3, SHIP construct reverts the SF-induced degranulation to that seen in SHIP^+/+ BMMCs.

(A) SHIP^+/+ and SHIP^−/− BMMCs expressing empty vector as well as SHIP^−/− BMMCs expressing the indicated constructs were treated with or without 400 ng/mL SF for 15 minutes and the degree of degranulation determined by measuring the release of β-hexosaminidase. Unstimulated values were subtracted for each cell type (typically <  10% degranulation) and the mean ± SE of duplicate determinations are shown. (B) SHIP^+/+ BMMCs and SHIP^−/− BMMCs expressing WT and T1 SHIP were treated with and without 200 ng/mL SF for 3 minutes and plasma membranes prepared as described in “Materials and methods.” Western analysis of the NP40 solubilized membranes with anti-SHIP (for SHIP^+/+ cells) or anti-HA (for SHIP^−/− BMMCs expressing the constructs) antibodies (upper panel) was followed by reprobing with anti-c-kit antibodies (lower panel) to confirm equal loading of each set of unstimulated and SF-stimulated lanes. Similar results were obtained in 2 separate experiments.