Figure 1.
Figure 1. Effect of SHP-1 on Stat-5 activation. SHP-1 negatively regulates Stat5 activation independently of the phosphatase activity. (A) Schematic representation of SHP-1 wild type and its mutants. (B) The 293 cells were cotransfected with expression plasmids encoding PRLR, Stat5, β-casein gene promoter/luciferase reporter construct, and either vector, SHP-1WT, SHP-1CS, or SHP-1δC. Cells were starved and left unstimulated or stimulated with ovine PRL (oPRL) (1 μg/mL) overnight. Luciferase activity was assayed, and data were presented as relative light units. Each bar represents the mean ± standard deviation values of triplicate samples. The luciferase activity was normalized to the expression of β-galactosidase. □ indicates absence, and ▪, presence, of oPRL. (C) Differentiated HC11 cells were transfected with either vector alone or expression plasmids for the indicated forms of SHP-1. Cells were starved and stimulated with oPRL for 24 hours. Total cell lysates were immunoblotted with a polyclonal antibody to milk proteins (upper panel). The membrane was stripped and reblotted with polyclonal antibody to SHP-1 (lower panel). (D) The 293 cells were transiently cotransfected with expression vectors for PRLR, Stat5, and either vector or the indicated forms of SHP-1. Serum-starved cells were left untreated or treated with oPRL (1 μg/mL) for 10 minutes. Total cell lysates were analyzed by immunoblot with the use of specific antibodies directed against the phosphorylated form of Stat5 (upper panel). The membrane was stripped and reprobed with monoclonal antibody to Stat5 (middle panel), then with polyclonal antibody to SHP-1 (lower panel). (E) The 293 cells were transiently cotransfected with expression vectors for the EPOR, Stat5, and either vector or the indicated forms of SHP-1. Serum-starved cells were left untreated or treated with EPO (5 U/mL) for 10 minutes. Total cell lysates were immunoprecipitated with polyclonal antibody to Stat5 and immunoblotted with monoclonal antibody to phospho-Stat5 (upper panel). Total cell lysates from the same transfection were immunodetected with monoclonal antibody to Stat5 (middle panel), then with polyclonal antibody to SHP-1 (lower panel). (F) Nb2 rat pre–T lymphoma cells and Nb2 cells stably expressing HA-tagged forms of SHP-1WT and SHP-1CS were starved for 18 hours and then left untreated or treated with oPRL (1 μg/mL) for 10 minutes. Cell lysates were immunoblotted with a monoclonal antibody to phospho-Stat5 (upper panel), a monoclonal antibody to Stat5 (middle panel), and a monoclonal antibody to HA tag (lower panel). (G) T47D cells were transfected with either vector or expression plasmids for the indicated forms of HA-tagged SHP-1. Cells were serum starved for 18 hours and then stimulated with hPRL (1 μg/mL) for 10 minutes. Cells were lysed, and immunoprecipitations were performed with polyclonal antibody to Stat5, followed by immunoblotting with a monoclonal antibody to phospho-Stat5 (upper panel). The membrane was stripped and reblotted with a monoclonal antibody to Stat5 (middle panel), then with a monoclonal antibody to HA tag (lower panel).

Effect of SHP-1 on Stat-5 activation. SHP-1 negatively regulates Stat5 activation independently of the phosphatase activity. (A) Schematic representation of SHP-1 wild type and its mutants. (B) The 293 cells were cotransfected with expression plasmids encoding PRLR, Stat5, β-casein gene promoter/luciferase reporter construct, and either vector, SHP-1WT, SHP-1CS, or SHP-1δC. Cells were starved and left unstimulated or stimulated with ovine PRL (oPRL) (1 μg/mL) overnight. Luciferase activity was assayed, and data were presented as relative light units. Each bar represents the mean ± standard deviation values of triplicate samples. The luciferase activity was normalized to the expression of β-galactosidase. □ indicates absence, and ▪, presence, of oPRL. (C) Differentiated HC11 cells were transfected with either vector alone or expression plasmids for the indicated forms of SHP-1. Cells were starved and stimulated with oPRL for 24 hours. Total cell lysates were immunoblotted with a polyclonal antibody to milk proteins (upper panel). The membrane was stripped and reblotted with polyclonal antibody to SHP-1 (lower panel). (D) The 293 cells were transiently cotransfected with expression vectors for PRLR, Stat5, and either vector or the indicated forms of SHP-1. Serum-starved cells were left untreated or treated with oPRL (1 μg/mL) for 10 minutes. Total cell lysates were analyzed by immunoblot with the use of specific antibodies directed against the phosphorylated form of Stat5 (upper panel). The membrane was stripped and reprobed with monoclonal antibody to Stat5 (middle panel), then with polyclonal antibody to SHP-1 (lower panel). (E) The 293 cells were transiently cotransfected with expression vectors for the EPOR, Stat5, and either vector or the indicated forms of SHP-1. Serum-starved cells were left untreated or treated with EPO (5 U/mL) for 10 minutes. Total cell lysates were immunoprecipitated with polyclonal antibody to Stat5 and immunoblotted with monoclonal antibody to phospho-Stat5 (upper panel). Total cell lysates from the same transfection were immunodetected with monoclonal antibody to Stat5 (middle panel), then with polyclonal antibody to SHP-1 (lower panel). (F) Nb2 rat pre–T lymphoma cells and Nb2 cells stably expressing HA-tagged forms of SHP-1WT and SHP-1CS were starved for 18 hours and then left untreated or treated with oPRL (1 μg/mL) for 10 minutes. Cell lysates were immunoblotted with a monoclonal antibody to phospho-Stat5 (upper panel), a monoclonal antibody to Stat5 (middle panel), and a monoclonal antibody to HA tag (lower panel). (G) T47D cells were transfected with either vector or expression plasmids for the indicated forms of HA-tagged SHP-1. Cells were serum starved for 18 hours and then stimulated with hPRL (1 μg/mL) for 10 minutes. Cells were lysed, and immunoprecipitations were performed with polyclonal antibody to Stat5, followed by immunoblotting with a monoclonal antibody to phospho-Stat5 (upper panel). The membrane was stripped and reblotted with a monoclonal antibody to Stat5 (middle panel), then with a monoclonal antibody to HA tag (lower panel).

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