The C2 domain is required for end-product allosteric activation of SHIP1 and binding of AQX-MN100. (A) SHIP1 enzyme initial velocities were determined at the indicated concentration of inositol-1,2,4,5-tetrakisphosphate (IP₄) substrate. The equation Y = 49 + 150 / (1 + 10^{log(80 − ×) − 0.22}) describes the line with R² = 0.98. (B) The ability of product PI-3,4-P₂ (20 μM) or AQX-MN100 (3 μM) to activate wild-type (WT) and C2 domain–deleted (ΔC2) SHIP1 enzyme was determined at 30 μM IP₄. (C) Recombinant C2 domain was preincubated for 30 minutes at 23°C with 4.0 μM AQX-MN100 or EtOH control and allowed to bind to PI-3,4-P₂ immobilized on membrane strips in a protein overlay assay as described in “Materials and methods, PLO assays.” (D) Recombinant C2 domain (10 nM) was coated onto copper chelate (His-Tag) YSi SPA Scintillation Beads in the presence of 0.25% BSA. Beads were then incubated with 0.185 MBq (5 μCi) of [³H]-AQX-MN100, and the bead-associated radioactivity was measured as described in “Materials and methods.” Data are expressed as means (±SEM) and are representative of at least 3 independent experiments.