SHIP2 contains PtdIns(3,4,5)P₃ 5-phosphatase activity. (A) NP-40 lysates from M07 and K562 cells were immunoprecipitated with anti-SHIP2 antibodies in the presence or absence of antigenic peptide. SHIP1 and control preimmune immunoprecipitates from M07 were used for comparison. The immunoprecipitates were incubated with equal amounts of [³²P]PtdIns(3,4,5)-P₃ in 5-phosphatase assay buffer. After 30 minutes at 37°C, the phospholipids were extracted with chloroform/methanol and separated by TLC. Radioactive lipids were detected by autoradiography. (B) The immunoprecipitates were obtained as described in (A) and incubated for 30 minutes at 37°C with [³H]Ins(1,3,4,5)-P₄ in 5-phosphatase buffer and assayed for hydrolysis. Results are expressed as picomoles of hydrolysis product per 30 minutes per immunoprecipitate. A blank sample containing no proteins was assayed and subtracted as background. The results are representative of four independent experiments.