Need for SOCS-1 in SHP-1–mediated negative regulation of Stat5 activation downstream of PRLR. (A) (B) Parental (SOCS-1^+/+) (panel A) as well as SOCS-1^–/– (panel B) MEF cells were cotransfected with expression plasmids encoding PRLR, Stat5, and either vector alone or the indicated forms of SHP-1 with the use of FuGENE 6 transfection reagent. Serum-starved cells were left untreated or treated with PRL for 15 minutes. Total cell lysates were imunoblotted with monoclonal antibody to phospho-Stat5 (upper panels). The same lysates were immunoblotted with monoclonal antibody to Stat5 (middle panels) and polyclonal antibody to SHP-1 (lower panels). These immunoblots were chosen as representative of 6 independent experiments.