Figure 4. Critical role of CIS C-terminus in blocking STAT5 activation.

(A) STAT5 reporter assay using the EpoR Tyr⁴⁰² motif. HEK-293T cells were transiently co-transfected with plasmids encoding the EpoR Tyr⁴⁰² bait, various mutants of FLAG-tagged CIS and with the pGL3-β-casein-luciferase reporter. After transfection, cells were left untreated (NS) or were stimulated with Epo for 24 h. Luciferase activities were measured in triplicate. Data are expressed as the stimulated/NS ratio+S.D. (B) EMSA using the EpoR Tyr⁴⁰² motif. HEK-293 Flp-In cells were transiently co-transfected with plasmids encoding the EpoR Tyr⁴⁰² bait, various FLAG-tagged CIS mutants and STAT5B. Nuclear lysates were incubated with ³²P-labelled probe corresponding to a β-casein STAT5-binding site to reveal active STAT5 complexes. The EpoRy402F bait (*) was used as a negative control. (C) STAT5 reporter assay using the wildtype EpoR. HEK-293T cells were transiently co-transfected with plasmids encoding the EpoR, various mutants of FLAG-tagged CIS and with the pGL3-β-casein-luciferase reporter. After transfection, cells were left untreated (NS) or were stimulated with Epo for 24 h. Luciferase activities were measured in triplicate. Data are expressed as the stimulated/NS ratio+S.D.