Fig. 7. Immunodepletion of HeLa nuclear extract with 2C11 causes a specific inhibition of pre-mRNA splicing. (A) In vitro splicing reaction with a ß-globin RNA probe. Splicing is inhibited by pre-incubation of nuclear extract with 2C11 beads. This effect is blocked by pre-treatment of the antibody with GroPIns(4,5)P2 but not GroPIns. A schematic representation of the starting probe, intermediates and product of the splicing reaction is shown on the right. (B) The beads used in the immunodepletion experiment presented in (A) and an equivalent amount of untreated 2C11 beads were analysed by western blotting using anti-RNA Pol IIo (H5). (C) Splicing efficiency was quantified and expressed as the percentage of processed RNA in the samples versus the total, taking the mock-treated sample as 100%. Bars represent the s.e. of six experiments. (D) The inhibition of splicing seen on treatment of the nuclear extract with immobilised 2C11 is partially rescued by pre-treatment of 2C11 beads with GroPIns(4,5)P2 but not GroPIns. Results are expressed as in (C) and bars represent the s.e. of four experiments.
