FIG. 4.

Association/dissociation of RAF-2p48 and NP. A GST pull-down assay was carried out as described in Materials and Methods, and proteins were detected by Western blotting analysis with rabbit anti-vRNP antiserum. As a positive control, 20% of input vRNP was loaded (Input). (A) Association of NP and RAF-2p48. GST (lanes 3 and 5) or GST-p48 (lanes 2, 4, and 6) was fixed on glutathione-Sepharose beads and incubated for 1 h at 37°C in the presence of a 100-ng equivalent of vRNP (lanes 3 and 4) or mnRNP (lanes 5 and 6). (B) Dissociation of RAF-2p48–NP complexes. GST (lanes 1, 2, 8, 9, and 10) or GST-p48 (lanes 3 to 7) was fixed on glutathione-Sepharose beads and incubated with mnRNP (lanes 5 to 10) at 37°C for 1 h. Unbound proteins were washed out, and beads were further incubated in the presence of 200 ng (lanes 1, 3, 6, and 9) or 1 μg (lanes 2, 4, 7, and 10) of free RNA (53-mer Vwt).