CIS inhibits STAT5 activity in a reconstituted system in HEK293 cells. Upper graph, plasmids for the EPO receptor, STAT5, and luciferase-reporter gene (construct A) were introduced into HEK293 cells as described in the legend to Fig 5, in the presence of an expression vector carrying the CIS gene (pME-CIS)(+) or unrelated protein (pME-CCF18)(−). After a 24-hour incubation in the presence or absence of EPO (10 U/mL), luciferase activity in each transfection was measured. Lower gels, after transfection, cells were cultured for 24 hours in DMEM containing 10% FCS without EPO, then starved for 12 hours in the medium containing 0.5% FCS. Cells were stimulated with EPO (10 U/mL) for 10 minutes. Cell extracts were immunoprecipitated with anti-STAT5 antibody, then resolved by SDS-PAGE and probed with antiphosphotyrosine (anti-PY) or anti-STAT5 antibodies.