Figure 2.
Figure 2. Effect of SHP-1 C-terminal tyrosine residues on PRLR signaling. SHP-1 C-terminal tyrosine residues deliver the inhibitory effect of SHP-1 in PRLR signaling. (A) Nb2 cells (left panel) and Jurkat cells (right panel) were starved overnight and then either left untreated or treated with PRL for 5 minutes. Cell lysates were used for immunoprecipitation with a polyclonal antibody to SHP-1 followed by immunoblotting with a monoclonal antibody to phosphotyrosine (4G10). The membranes were stripped and reblotted with a polyclonal antibody to SHP-1 (lower panels). (B) Nb2 cells were starved overnight and left unstimulated or stimulated with PRL for 5 minutes (left panel). T47D cells were starved overnight and left unstimulated or stimulated with hPRL (1 μg/mL) and EGF (20 ng/mL) (as a positive control) for 5 minutes (right panel). Cell extracts were immunoprecipitated with a polyclonal antibody to SHP-1 and subjected to Western blotting analysis with a monoclonal antibody to Grb2 (upper panels). The membranes were stripped and reblotted with a polyclonal antibody to SHP-1 (lower panels). TL indicates total cell extracts. (C) The 293 cells were cotransfected with expression plasmids for PRLR, Stat5, and the indicated forms of SHP-1 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. (D) The 293 cells were transiently cotransfected with expression plasmids encoding PRLR, Stat5, and either vector or the indicated forms of SHP-1. Serum-starved cells were left unstimulated or stimulated with oPRL. Total cell lysates were probed with monoclonal antibody to phospho-Stat5 (upper panel), monoclonal antibody to Stat5 (middle panel), or polyclonal antibody to SHP-1 (lower panel). (E) Cell lysates from 293 cells overexpressing EPOR, Stat5, and either vector alone or the indicated forms of SHP-1 that were left unstimulated or stimulated with EPO (5 U/mL) were immunoprecipitated with polyclonal antibody to Stat5 and immunoblotted with monoclonal antibody to phospho-Stat5 (upper panel). The membrane was stripped and reblotted with monoclonal antibody to Stat5 (middle panel) or polyclonal antibody to SHP-1 (lower panel). (F) The 293 cells were transiently cotransfected with expression plasmids encoding PRLR and either vector alone or indicated forms of SHP-1 expression vectors. Serum-starved cells were left untreated or treated with PRL for 10 minutes. Cell lysates were immunoprecipitated with polyclonal antibody to SHP-1, and proteins were separated on SDS-PAGE and transferred to nitrocellulose membrane. Direct binding of GST-Grb2 fusion protein was assessed by far Western analysis (upper panels). The same membrane was stripped and reblotted with anti–SHP-1 (lower panels).

Effect of SHP-1 C-terminal tyrosine residues on PRLR signaling. SHP-1 C-terminal tyrosine residues deliver the inhibitory effect of SHP-1 in PRLR signaling. (A) Nb2 cells (left panel) and Jurkat cells (right panel) were starved overnight and then either left untreated or treated with PRL for 5 minutes. Cell lysates were used for immunoprecipitation with a polyclonal antibody to SHP-1 followed by immunoblotting with a monoclonal antibody to phosphotyrosine (4G10). The membranes were stripped and reblotted with a polyclonal antibody to SHP-1 (lower panels). (B) Nb2 cells were starved overnight and left unstimulated or stimulated with PRL for 5 minutes (left panel). T47D cells were starved overnight and left unstimulated or stimulated with hPRL (1 μg/mL) and EGF (20 ng/mL) (as a positive control) for 5 minutes (right panel). Cell extracts were immunoprecipitated with a polyclonal antibody to SHP-1 and subjected to Western blotting analysis with a monoclonal antibody to Grb2 (upper panels). The membranes were stripped and reblotted with a polyclonal antibody to SHP-1 (lower panels). TL indicates total cell extracts. (C) The 293 cells were cotransfected with expression plasmids for PRLR, Stat5, and the indicated forms of SHP-1 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. (D) The 293 cells were transiently cotransfected with expression plasmids encoding PRLR, Stat5, and either vector or the indicated forms of SHP-1. Serum-starved cells were left unstimulated or stimulated with oPRL. Total cell lysates were probed with monoclonal antibody to phospho-Stat5 (upper panel), monoclonal antibody to Stat5 (middle panel), or polyclonal antibody to SHP-1 (lower panel). (E) Cell lysates from 293 cells overexpressing EPOR, Stat5, and either vector alone or the indicated forms of SHP-1 that were left unstimulated or stimulated with EPO (5 U/mL) were immunoprecipitated with polyclonal antibody to Stat5 and immunoblotted with monoclonal antibody to phospho-Stat5 (upper panel). The membrane was stripped and reblotted with monoclonal antibody to Stat5 (middle panel) or polyclonal antibody to SHP-1 (lower panel). (F) The 293 cells were transiently cotransfected with expression plasmids encoding PRLR and either vector alone or indicated forms of SHP-1 expression vectors. Serum-starved cells were left untreated or treated with PRL for 10 minutes. Cell lysates were immunoprecipitated with polyclonal antibody to SHP-1, and proteins were separated on SDS-PAGE and transferred to nitrocellulose membrane. Direct binding of GST-Grb2 fusion protein was assessed by far Western analysis (upper panels). The same membrane was stripped and reblotted with anti–SHP-1 (lower panels).

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