Figure 6

PLZF decreases the dimerization of RARα with RXRα. A) PLZF decreases the interaction of RAR with RXR in intact cells. HeLa cells were transiently transfected with the UAS-tkLuc reporter gene, the pCMV-Gal4-RXRα and the pCMV-VP16-RARα expression vectors and the pSG5-PLZF or the pCMV-PLZF 3ZF as indicated. The pRSV-βGal expression was used as a control in this 2-hybrid assay. Cells were treated with 0.1 μM of the RARα-selective ligand Am580, and luciferase activity was assayed 16 h later as described in Figure 4. B) PLZF inhibits the dimerization of RARα with RXRα in-vitro. RXRα was synthesized in vitro as a ³⁵S-labeled protein, by coupled transcription/translation, in rabbit reticulocyte lysate and was incubated with increasing amounts of PLZF or of control protein (luciferase) as non-labeled proteins. Protein mixes were then incubated with a GST-RARα-Sepharose affinity matrix with 1 μM atRA or not. Proteins were then separated and quantified as indicated in Figure 1. Representative autoradiographs are shown. Bar graphs show data averaged from 2 independent experiments.