Figure 3.

FL cells transduced with Tyr577Pheβ_c show enhanced Gab2 tyrosine phosphorylation in the absence of Shc phosphorylation. (A) FL cells transduced with either wtβ_c or Tyr577Phe were cultured for 12 hours in RPMI containing 0.5% HI-FCS and stimulated with GM-CSF for 5 minutes prior to lysis. Cell lysates were cleared and immunoprecipitated with antibodies to β_c, Shc, SHP2, or Gab2. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the antibodies as shown in Figure 1A. (B) Mutation of Tyr577 does not abolish SHP2 coimmunoprecipitation with β_c. CTL-EN cells expressing either wtβ_c or Tyr577Phe were factor deprived for 12 hours before stimulation with 50 ng/mL GM-CSF for 1, 2, 5, 15, 30, or 45 minutes. Following stimulation, β_c was immunoprecipitated with the anti-β_c antibody 1C1, and the immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-phosphoTyr577, antiphosphotyrosine 4G10, anti-β_c, or anti-SHP2 antibodies. A vertical line has been inserted to indicate where a gel lane was cut. These were from separate gels run from a single experiment. (C) FL cells transduced with either wtβ_c or Tyr577Phe were cultured for 12 hours before stimulation with 50 ng/mL GM-CSF for 5, 15, or 30 minutes. Following stimulation, the cells were lysed, and the lysates were subjected to SDS-PAGE and immunoblotted with phosphospecific antibodies to JAK2, STAT5A, Akt, and Erk. An anti-Erk antibody was used as a loading control. The data shown is representative of 3 separate experiments performed using transduced FL cells.