IL-2–dependent WT1 binding to the hTERT promoter. (A) The hTERT-3915 promoter was biotin labeled and bound to streptavidin-agarose beads, followed by incubation with 1185 cellular extracts from cells cultivated overnight in the presence or absence of IL-2. Bound proteins were visualized using anti-WT1 or anti–c-Myc. The percentage of increased binding of WT1 to the hTERT promoter was calculated by spot densitometry as indicated, with 1185 cells grown in the presence of IL-2 considered to be 100%. (B) 293T cells were transfected with wild-type WT1 (SS) or mutant WT1 (FF), along with the hTERT-Luc promoter, or wild-type WT1 (SS) or mutant WT1 (FF), along with the hTERT-Mut-Luc promoter (defective for WT1 binding). Extracts were assayed 48 hours after transfection for luciferase activity. Cell extracts were probed with anti-WT1 and antiactin to verify expression. Average luciferase values were as follows: hTERT-Luc promoter (150 518), + WT1 (SS) (79 578), + WT1 (FF) (241 727) (SD measured from at least 2 independent experiments) and for the hTERT-Mut Luc promoter (1 166 792), + WT1 (SS) (1 468 345), + WT1 (FF) (1 736 347) (SD measured from multiple readings from the same experiment).