ATRA binding and transcriptional activation by a PML/RARα mutant depend on the cell line in which the receptor is expressed. (A) Comparison of ATRA-binding activity of PML/RARα expressed by NB4 cells (○) with the PML/RARα mutant I410T expressed by NB4-MRA1 (•) cells. Nuclear extracts of NB4 and NB4-MRA1 cells were subjected to Western blot analysis. The left-pointing arrow indicates expression of the 110-kDa PML/RARα wild-type or mutant I410T. (B) ATRA-binding activity of PML/RARα (•) and PML/RARα mutant I410T (○) overexpressed in Cos-1 cells. Nuclear extracts were incubated with 10 nM [³H]-ATRA or with the addition of 200-fold excess unlabeled ATRA (▪) and subjected to HPLC analysis using a 6 HR 10/30 size exclusion column. Down-pointing arrows indicate approximate retention times of void volume, PML/RARα multimers (670 kDa), and RARα monomers (50 kDa). Nuclear extracts of Cos-1–transfected cells were subjected to Western blot analysis for PML/RARα wild-type or mutant I410T (left-pointing arrow). (C) Comparison of the ligand-dependent transcriptional activity, on a DR5-tk-CAT, of PML/RARα wild-type and mutant I410T endogenously expressed in APL cells and overexpressed in Cos-1 cells. Assays were performed in triplicate and repeated at least 3 times. Bars represent standard deviation of the mean.