Pi 3 4 5 p3 dic8

Phosphatase activity was assessed by monitoring dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5-P3) similarly as described before [13 (link), 36 (link)]. Reaction mixture (25 μL) consisted of 100 mM Tris (pH 8.0), 10 mM DTT, 200 μM PI-3,4,5-P3 (diC8; Echelon, U.S.A.) and varied amounts of CdtB or CdtB210. Human recombinant PTEN (Cayman Chemicals, U.S.A.) was used as a control enzyme hydrolyzing PI-3,4,5-P3. Hydrolysis was carried out at 37°C for 30 min. Reaction was terminated by the addition of 15 μL of 100 mM N-ethylmaleimide (Sigma-Aldrich). Inorganic phosphate levels were then measured using malachite green solution (Malachite Green Assay Kit; Echelon); 35 μL of reaction mixture was combined with 100 μL of the malachite green solution for 20 min at room temperature. Absorbance at 620 nm was measured and phosphate release was quantified by comparison to inorganic phosphate standards (Echelon, U.S.A.). Data are means ±SEM of triplicates.

The catalytic activities of the recombinant SHIP2 and SHIP1 phosphatase domains, and SHIP2 immunoprecipitated from cell and tissue lysates were determined by malachite green phosphate assay using PI(3,4,5)P3 diC8 at the final concentration of 100 µM (Echelon Biosciences, Salt lake City, UT, USA) as previously described 17 (link).