Reconstitution of CIS promoter activation by STAT5 and the EPO receptor in HEK293 cells. HEK293 cells were transfected with CIS promoter-luciferase reporter constructs in combination with the EPO receptor and/or STAT5 cDNAs in pXM expression vector. MGF+ means A construct carrying wild-type promoter and MGF− means D construct containing mutant promoter lacking all four MGF boxes, as shown in Fig 4. In minus (−), same amount of pXM plasmid without insert cDNA was added. Twenty-four hours after transfection, cells were stimulated with or without EPO (10 U/mL) and cell extracts were analyzed for luciferase activity.