PLZF is a negative regulator of retinoic acid receptor transcriptional activity

Additional File 1:

PLZF and ligand interaction with RARα. A) PLZF decreases the dimerization of RAR with RXR in a ligand structure-independent manner. RXR-RAR interaction was tested by GST pulldown assays as in Figure 6B in the presence of RAR agonists (atRA, CD367, Am580) or of an antagonist (Ro41-5253). B) The interaction of PLZF with RAR does not alter RAR ligand binding affinity. Ligand binding assays were carried out as follows: The GST-RARα was expressed as described in the Materials and Methods section. PLZF or a control protein were synthesized with the Quick T7 TnT kit (Promega) with unlabeled methionine. GST-RARα was incubated on a rotating wheel for 2 h at 4°C with increased amounts of PLZF or of control protein in the presence of 20 nM [³H] atRA (50 Ci/mmol, PerkinElmer), and with or without 1 μM unlabeled atRA to assess non specific binding. Conditions were strictly similar to those used in GST pulldown experiments. 20 μL of 50% Sepharose-GSH were then added and incubated on a rotating wheel for 1 h at 4°C. Unbound material was removed by three successive washes of Sepharose beads with 200 μL of ice-cold 1 × PBS. The amount of [³H] bound to the matrix was determined by liquid scintillation counting. Results are plotted as a function of atRA concentration. They demonstrate that the ligand binding activity of RARα is not altered in the presence of either PLZF or a control protein (luciferase).

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