Figure 5.

Mutation of Tyr577Phe in β_c abolishes the hydrolysis of PI-(3,4,5)P3 in response to GM-CSF. CTL-EN cells expressing either wtβ_c or Tyr577Phe were factor deprived for 12 hours in RPMI containing 0.5% HI-FCS before stimulation with either 0 or 50 ng/mL GM-CSF. Following stimulation, the cells were lysed, and the lysates were cleared and subjected to immunoprecipitation with anti-Shc antibody or anti-β_c antibody. (A) Shc immunoprecipitates were subjected to SDS-PAGE and immunoblotting with antibody to phospho-SHIP. (B) β_c immunoprecipitates subjected to a PtdIns(3,4,5)P₃ 5-phosphatase assay. (C) Quantification of PtdIns(3,4,5)P₃ 5-phosphatase activity in each immunoprecipitate. The bars represent the mean (± SD) from 2 independent experiments.