Effect of ATRA on interactions STAT5b-RARα with GST-SMRT and on STAT5b-RARα stability.
Radiolabeled wild-type or mutant STAT5b-RARα was incubated with GST-SMRT in the presence of the indicated concentrations of ATRA. After absorption with glutathione Sepharose, the proteins were separated and analyzed by autoradiography (A) and PhosphorImager analysis (B). COS-7 cells (C) and HeLa cells (D) were transiently transfected with STAT5b-RARα or PLZF-RARα and incubated without (lanes 1 and 3) or with (lanes 2 and 4) 10−6 M ATRA for 24 hours. Cells were lysed; proteins were separated by SDS-PAGE and immunoblotted with RARα antibody (upper panel) as well as β-actin antibody (bottom panel), which was used as loading control. Densitometry analysis is presented below each immunoblot. The results shown are representative of up to 3 separate experiments. The empty triangle to the right of each upper panel indicates presumed proteolytic fragments of PLZF-RARα.
