Fig. 2. Immunodepletion of CDC5L in HeLa nuclear extract inhibits pre-mRNA splicing. (A) Supernatants from immunodepletion experiments were probed by western blotting using anti-CDC5L antibody and the protein revealed using ECL (Amersham). Lanes 1 and 2 are duplicates containing supernatants of mock immunodepletions using pre-immune IgG. Lane 3 contains the supernatant from anti-CDC5L antibody immunodepleted nuclear extract and lane 4 is similar to lane 3 except that the antibody was pre-incubated with specific peptide before use in the immunodepletion experiment. The arrow indicates the CDC5L bands. (B) Proteins from the beads corresponding to the immunodepletions in (A) were separated by SDS–PAGE, transferred to nitrocelluose and probed with anti-CDC5L antibody as above. Lane numbers are identical to (A) except that the samples in each lane contain protein immunoprecipitated onto beads by bound antibody. The arrowhead indicates the CDC5L band while the arrow (Ab) shows bands corresponding to antibody heavy chain polypeptides. CDC5L is only detected in lane 3. (C) The supernatants in (A) were used in pre-mRNA splicing reactions, and the splicing intermediates and products separated on a 10% polyacrylamide–8 M urea denaturing gel. The symbols on the right of the figure represent the pre-mRNA and the different splicing intermediates and products. Bands not marked by symbols correspond to partial degradation products of the pre-mRNA. Lane 1 contains a pre-mRNA splicing control using untreated nuclear extract. Lanes 2 and 3 are duplicates from mock depletion experiments. The lanes marked 4 and 5 contained duplicates of splicing reactions using nuclear extracts depleted of CDC5L, while lanes 6 and 7 contain splicing reactions using nuclear extract treated with anti-CDC5L antibody pre-blocked with the antibody specific peptide. (D) S100 extract restores splicing activity to nuclear extract depleted with anti-CDC5L antibodies. Lane 1 shows a splicing reaction with untreated nuclear extract. Lane 2 contains a mock depletion using sheep pre-immune IgG. Lanes 3 and 4 show duplicate pre-mRNA splicing reactions using nuclear extracts immunodepleted with anti-CDC5L antibodies, and lanes 5 and 6 represent duplicate splicing reactions using nuclear extracts immunodepleted with anti-CDC5L antibodies to which have been added ∼16–20 µg of S100 cytoplasmic extract.