Fig 5.

BN82685 is a general splicing inhibitor that acts after the first catalytic step. (A) In vitro splicing reaction mixtures containing biotin-labeled Ad2, CDC14-15, or IgM C3-C4 pre-mRNA substrates in the presence of DMSO or 10 μM BN82685 were measured by the EJIPT assay using anti-Y14 antibody. (B) Mixtures described in panel A with ³²P-labeled pre-mRNAs spliced in vitro were analyzed on denaturing PAGE gels. The identities of splicing intermediates for Ad2 and CDC14-15 are shown on the left, and intermediates for IgM C3-C4 are shown on the right. (C) Compounds were added to splicing reaction mixtures with CDC14-15 pre-mRNA at the final indicated concentrations. Phosphorimaging quantification of each intermediate on the splicing gel is expressed as a percentage compared to DMSO after normalizing to total counts in the lane (below).