FIG. 4.
Competition for TAP binding between CTE and TBE. (A) Pulldown assays with GST-TAP61-610 were performed using 32P-labeled CTE of SRV-1 in the absence of heparin (left panel) or in the presence of 0.5 μg of heparin per ml (middle panel) or 2 μg of heparin per ml (right panel). CTESRV−1 RNA (open rectangles), TBE RNA (filled rectangles), and random RNA from the unselected genomic library (closed diamonds) were used as unlabeled competitors. The fraction of 32P-labeled CTE RNA bound to TAP was plotted against the final concentration of unlabeled competitor RNAs. (B) Gel mobility shift assays with GST-TAP61-610 were performed using binding conditions that were the same as those used to obtain the results shown as in panel A, in the presence of 0.5 μg of heparin per ml. The unlabeled competitor RNAs were present at 333 nM, as indicated. The complexes (bound) and the probe (free) were separated on 1% agarose gels and detected by autoradiography. Similar results were obtained in three independent experiments.