Figure 8.

 Specific binding of SF2/ASF to an SF2/ASF-selected ESE. (A) UV cross-linking competition binding assay. Radiolabeled exon M2 RNA (20 fmoles) comprising the B1 winner sequence (B1E) was incubated under splicing conditions in HeLa nuclear extract. Subsequent UV cross-linking and RNase digestion resulted in label transfer predominantly to two proteins of 34 and 50 kD. The former, which binds specifically, is presumed to be SF2/ASF (see below). Cold competitor RNAs containing either the B1 winner insert, an SRp55-selected insert (C4E), an SRp40-selected insert (E7E), or other control sequence inserts, were present in excess, as indicated above the autoradiograph. (Lane 1) No competitor; in the remaining lanes, the indicated competitors were present in 5-fold excess (even lanes) or 50-fold excess (odd lanes) over the labeled B1E RNA. (B) Immunoprecipitation of SF2/ASF UV cross-linked to the B1E RNA. UV cross-linking was carried out as in A, lane 1. A 5% equivalent of the input was loaded directly (lane 1). Parallel reactions were incubated with a control antibody (lane 3), or with anti-SF2/ASF monoclonal antibody (lane 2), and the immunoprecipitates were recovered in SDS gel loading buffer. In A and B, the samples were analyzed by SDS-PAGE and autoradiography.