FIG. 4.

DAX-1 binds to RNA. (A) RNA homopolymer binding assay. Binding to agarose beads coupled to poly(A), poly(C), poly(G), and poly(U) is shown for human (lanes 2 to 5) and mouse (lanes 7 to 10) DAX-1 and for luciferase (Luc) (lanes 12 to 15) translated in a rabbit reticulocyte (ret.) lysate. Binding to RNA homopolymers is also shown for DAX-1 translated in a wheat germ system (lanes 17 to 20) and expressed in insect cells (lanes 22 to 25). A 1:10 ratio of the input protein is shown in each case (lanes 1, 6, 11, 16, and 21). The assay was performed in a buffer containing 0.25 M NaCl. ³⁵S-labeled proteins were detected by fluorography, and DAX-1 expressed in a baculovirus system was detected by Western blot. (B) Salt sensitivity of the binding of DAX-1 to poly(A) (lanes 2 to 5), poly(C) (lanes 6 to 9), poly(G) (lanes 10 to 13), and poly(U) (lanes 14 to 17) was tested in buffers containing NaCl concentrations of 0.1, 0.25, 0.5, and 1 M. Lane I, 1:10 of the input protein (lane 1). (C) Northwestern binding assay using a ³²P-labeled 240-nucleotide riboprobe transcribed from PvuII-linearized pBluescript and proteins immobilized on a nitrocellulose membrane. The Ponceau red-stained membrane is also shown on the left. Lanes show molecular size markers (lane 1), BSA (10 μg, lane 2), and DAX-1 (10 μg, lane 3).