Figure 4.
Figure 4. Grb2 and SHP-1 regulation of Jak2/SOCS-1 association. (A) 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 stimulated with PRL for the times indicated. Cell lysates were used for immunoprecipitations with polyclonal antibody to Grb2 and immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (upper panel). The same membrane was stripped and immunoblotted with monoclonal antibody to Grb2 (lower panel). (B) The 293 cells were cotransfected with expression vectors encoding PRLR, myc–SOCS-1, and the indicated forms of SHP-1. Serum-starved cells were left untreated or stimulated with PRL for 15 minutes. Cell lysates were immunoprecipitated with polyclonal antibody to SHP-1 and immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (upper panels). The same membranes were stripped and reblotted with polyclonal antibody to SHP-1 (lower panels). (C) Cell lysates from 293 cells transfected with the same expression vectors as in panel A were stimulated with PRL for different times, immunoprecipitated with polyclonal antibody to Jak2, and then immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (upper panel). The same membrane was stripped and immunoblotted with polycclonal antibody to Jak2 (lower panel). (D) Parental Nb2 cells as well as Nb2 cells stably overexpressing SHP-1WT and SHP-1CS were serum starved overnight and then left unstimulated or stimulated with PRL for 1 hour. Cell lysates were immunoprecipitated with polyclonal antibody to SOCS-1 and immunoblotted with polyclonal antibody to Jak2 (upper panel). Total cell lysates were immunoblotted with polyclonal antibodies to SOCS-1 (middle panel) and Jak2 (lower panel). (E) The 293 cells were transfected with the same expression vectors as panel B. Cells were starved overnight and stimulated with PRL for different times. Cell lysates were immunoprecipitated with polyclonal antibody to Jak2 and immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (upper panels). The same membranes were stripped and reblotted with polyclonal antibody to Jak2 (lower panels). Total cell lysates from the same transfections were immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (middle panels).

Grb2 and SHP-1 regulation of Jak2/SOCS-1 association. (A) 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 stimulated with PRL for the times indicated. Cell lysates were used for immunoprecipitations with polyclonal antibody to Grb2 and immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (upper panel). The same membrane was stripped and immunoblotted with monoclonal antibody to Grb2 (lower panel). (B) The 293 cells were cotransfected with expression vectors encoding PRLR, myc–SOCS-1, and the indicated forms of SHP-1. Serum-starved cells were left untreated or stimulated with PRL for 15 minutes. Cell lysates were immunoprecipitated with polyclonal antibody to SHP-1 and immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (upper panels). The same membranes were stripped and reblotted with polyclonal antibody to SHP-1 (lower panels). (C) Cell lysates from 293 cells transfected with the same expression vectors as in panel A were stimulated with PRL for different times, immunoprecipitated with polyclonal antibody to Jak2, and then immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (upper panel). The same membrane was stripped and immunoblotted with polycclonal antibody to Jak2 (lower panel). (D) Parental Nb2 cells as well as Nb2 cells stably overexpressing SHP-1WT and SHP-1CS were serum starved overnight and then left unstimulated or stimulated with PRL for 1 hour. Cell lysates were immunoprecipitated with polyclonal antibody to SOCS-1 and immunoblotted with polyclonal antibody to Jak2 (upper panel). Total cell lysates were immunoblotted with polyclonal antibodies to SOCS-1 (middle panel) and Jak2 (lower panel). (E) The 293 cells were transfected with the same expression vectors as panel B. Cells were starved overnight and stimulated with PRL for different times. Cell lysates were immunoprecipitated with polyclonal antibody to Jak2 and immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (upper panels). The same membranes were stripped and reblotted with polyclonal antibody to Jak2 (lower panels). Total cell lysates from the same transfections were immunoblotted with monoclonal antibody to myc tag detecting SOCS-1 (middle panels).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal