Figure 9. Effect of U6atac distal 3′ SL mutations on p65-C-RRM binding.

(a) EMSA of WT and mutant U6atac distal 3′ SL (nts. 91-109) oligonucleotides with GST-p65-C-RRM. The location of the respective mutations in the U6atac SL RNA oligonucleotides are illustrated in Fig. 8a. WT U6atac distal 3′ SL ³²P-labeled oligonucleotides were incubated with 0 (lane 1), 20, (lane 2) 40 (lane 3) and 60 (lane 4) nmoles of GST-p65-C-RRM. ³²P-labeled oligonucleotides were incubated with (+) or without (-) GST-p65-C-RRM (40 nmoles). RNA–protein complexes were resolved on a 6% native polyacrylamide gel. Arrows on the right denotes the position of the RNA-protein complex band and free RNA. The upper and lower parts of the gel represent different exposures. (b) Quantitation of the RNA-protein complex formed with 40 nmoles of GST-p65-C-RRM in the gel shown in (a). The RNA-protein complex for the WT corresponds to lane 3 in (a).