Stat5 is likely not an immediate early activator of the βRARE.
(A) SCF-dependent EML cells were incubated with IL-3 for the indicated period of time, and the cells were then electroporated with the βRARE-tk-Luc reporter (25 μg). Five hours later, the cells were harvested and relative luciferase activity was determined on cell extracts using a β-galactosidase reporter (20 μg) as an internal control. (B) EML cells as well as EML cells treated overnight with IL-3 as indicated were electroporated with 25 μg of the p(UAS)5-LUC reporter together with 25 μg of each of the indicated plasmids and then cultured in the presence or absence of ATRA (1 μM) as indicated. After 5 hours the relative luciferase activity was determined on cell extracts using a β-galactosidase reporter (20 μg) as an internal control. For panels A and B, results represent the averages and SDs of triplicate samples. As detailed in “Materials and methods” the GAL-RARα and GAL-RARΔN vectors harbor the full-length and N-terminal–deleted RARα cDNA, respectively, whereas the GAL-RARΔNΔC vector also has a deleted C-terminal domain, which renders it unresponsive to ATRA stimulation. (C) BaF3-RXR cells were cultured in IL-3–deficient media for 24 hours. Nuclear extracts from these cells as well as from these same cells stimulated for 1 hour with IL-3 as indicated were used in an EMSA and EMSA supershift with the indicated oligonucleotides and antibody. The actin promoter fragment binds to a ubiquitously expressed nuclear protein23 and thus serves as a control for the amount of nuclear extract used in the EMSA.
