Effect of Grb2 on cytokine-induced Stat5 activation. Grb2 is a negative regulator of cytokine-induced Stat5 activation. (A) The 293 cells were cotransfected with expression plasmids for PRLR, Stat5, and the indicated forms of Grb2 along with β-casein gene promoter/luciferase reporter construct. A luciferase assay was performed, and luciferase activity was normalized to the relative β-galactosidase values. Results represent means and standard deviations of 3 independent experiments. □ indicates absence, and ▪, presence, of oPRL. (B) The 293 cells were transiently cotransfected with expression plasmids encoding PRLR, Stat5, and either vector alone or the indicated forms of Grb2. Following 18 hours of serum starvation, cells were left unstimulated or stimulated with PRL for 10 minutes. Cell lysates were immunodetected by means of monoclonal antibodies to phospho-Stat5 (upper panel), Stat5 (middle panel), or Grb2 (lower panel). (C) The 293 cells cotransfected with expression plasmids encoding the EPOR, Stat5, and either vector alone or the indicated forms of Grb2. Following overnight starvation, cells were left unstimulated or stimulated with EPO (5 U/mL) for 10 minutes. Cell lysates were immunoprecipitated with polyclonal antibody to Stat5 and immunoblotted with monoclonal antibody to phospho-Stat5 (upper panel). The membrane was stripped and reprobed with monoclonal antibodies to Stat5 (middle panel) or Grb2 (lower panel). (D) T47D cells were transfected with either vector alone or the indicated forms of HA-tagged Grb2 expression plasmids. Serum-starved cells were stimulated with hPRL (1 μg/mL) for 10 minutes and lysed. Immunoprecipitations were performed with polyclonal antibody to Stat5, followed by immunoblotting with monoclonal antibody to phospho-Stat5 (upper panel). The membrane was stripped and reprobed with monoclonal antibodies to Stat5 (middle panel) or HA tag (lower panel).