Enhanced and prolonged PI3K activation in CTL-EN cells expressing β_c Tyr577Phe 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 50 ng/mL GM-CSF for 0, 15, 30, or 60 minutes. The cells were then lysed, and the lysates cleared and subjected to immunoprecipitation with anti-β_c antibodies. (A) Immunoprecipitates were then tested in an in vitro kinase assay. (B) Quantification of counts from panel A as measured in a liquid scintillation counter. Experiment shown is representative of 3 separate assays that were performed. (C) FL cells transduced with either wtβ_c or Tyr577Phe were stimulated with GM-CSF for 0, 5, 15, 30, 60, 120, 240, or 360 minutes before lysis. Cell lysates were cleared and immunoprecipitated with antibodies to β_c. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the antibodies as shown. Anti-β_c antibody 1C1 and anti-p85 were used as a loading controls for immunoprecipitations and lysates, respectively.