FIGURE 3.

SHIP1 is phosphorylated in HEK-293 cells. HEK-293 cells were transfected with SHIP1, labeled with ³²P, and stimulated with 100 μm Sp-cAMPS (Sp) for 15 min or 10 μm isoproterenol (Iso) for 5 min. The FLAG-tagged SHIP1 was immunoprecipitated from cell extracts, resolved on a SDS gel, and subjected to autoradiography. A, upper panel presents the autoradiograph showing the effect of Sp-cAMPS or isoproterenol on phosphorylation of SHIP1. The lower panel presents the amount of ³²P incorporated into SHIP1 based on the integrated band intensity normalized for protein expression. The data are expressed as percentages of control (Ctr). *, p < 0.05 (n = 3). B, treating cells with Sp-cAMPS or isoproterenol stimulates the activity of immunopurified SHIP1. HEK-293 cells were transfected with SHIP1, and the cells were incubated with 100 μm Sp-cAMPS for 15 min or 10 μm isoproterenol for 5 min before lysis. Both Sp-cAMPS and isoproterenol significantly increased activity. ***, p < 0.001 (n = 6).