FIG. 5.

Analysis of ASAP-S, Acinus-L, and SAP18 in the in vitro splicing assay. Increasing amounts of purified ASAP-L (1.25 and 5 μl; lanes 5, 6, 15, and 16), ASAP-S (0.875 μl and 3.5 μl; lanes 7, 8, 17, and 18), and baculovirus-expressed recombinant Acinus-L (30, 90, and 260 ng; lanes 2, 3, 4, 12, 13, and 14) or SAP18 (4 and 12 ng; lanes 9, 10, 19, and 20) were added to S100 extracts complemented with limiting (5 ng; lanes 1 to 10) or optimal (50 ng; lanes 11 to 20) amounts of ASF/SF2. Lane 1, S100 plus 5 ng of ASF/SF2 only; lane 11, S100 plus 50 ng of ASF/SF2 only. As determined by Western blot analyses (data not shown), 5 μl of purified ASAP-L contained amounts of Acinus-L and SAP18, which were stoichiometric to 260 ng of recombinant Acinus-L and 12 ng of recombinant SAP18, respectively. Purified ASAP-S (3.5 μl) contained stoichiometric amounts of ASAP to 5 μl of ASAP-L. RNA was fractionated on a denaturing polyacrylamide gel, and splicing products, indicated schematically on the right, were visualized by autoradiography.