Fig. 5. Purified CDC5L-associated complex restores splicing activity to nuclear extract depleted of the protein. 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. (A) About 0.2 µg of eluate from the anti-CDC5L affinity column was added back to the immunodepleted extract before the extracts were used in pre-mRNA splicing reactions. The pre-mRNA splicing intermediates and products were separated on a 10% polyacrylamide–8 M urea denaturing gel. Lane 1 contained ∼15% of the input pre-mRNA. Lanes 2 and 3 contained untreated and mock-depleted nuclear extracts, respectively. Lanes 4 and 5 contain duplicate splicing reactions using nuclear extract immunodepleted using anti-CDC5L antibodies. Lanes 6 and 7 have duplicate splicing reactions using the same nuclear extract as above except that these lanes also have the CDC5L-containing eluate from the affinity column. Lane 9, marked CTRL, contains a control splicing reaction using immunodepleted nuclear extract as above except that here the eluate from the antibody specific peptide pre-blocked column (see Figure 4B, lane 1) was used in the add back. (B) Fractions from the Mono-Q and Mono-S columns were used to restore splicing activity to CDC5L immunodepleted nuclear extract. Lane 1 contains ∼15% of the input pre-mRNA used in the splicing reaction. Lanes 2 and 3 have untreated and mock depleted nuclear extracts, respectively. Lane 4 contains a splicing reaction using nuclear extract immunodepleted using anti-CDC5L antibodies. The same nuclear extract was used for the splicing reactions in lanes 5–12. In the add back experiments, the reactions in lanes 5–7 also had 0.5, 1 and 2 µl, respectively, of the pooled Mono-Q fractions containing CDC5L and lanes 8–10 had similar amounts of the pooled CDC5L-containing Mono-S fractions, respectively. Lane 11 is a control add back experiment containing a pool of Mono-Q fractions 16 and 19 that lack CDC5L, while lane 12 contains a pool of Mono-S column fractions 19 and 22 that is deficient in CDC5L (see Figure 4C above).