FIG. 1.

Characterization of HeLa cell lines stably expressing FLAG/HA-tagged AD002 and SPF27. (A) Nuclear extract was prepared from FLAG/HA-AD002, FLAG/HA-SPF27, or control cell lines as indicated above. Proteins were recovered from increasing amounts of nuclear extract (as indicated above each lane) and separated by SDS-PAGE. Proteins were transferred to nitrocellulose and further analyzed by Western blotting with anti-FLAG (lanes 1 to 12, middle panels), anti-AD002 (lanes 1 to 6, upper panel), and anti-SPF27 antibodies (lanes 7 to 12, upper panel). For a loading control, all membranes were also probed with anti-61K antibodies (lanes 1 to 12, bottom panel). The position of the endogenous and the FLAG/HA-tagged protein is indicated on the right of each panel. Note that in the middle panel, the anti-FLAG-SPF27 signal does not increase when more than 2.5 μl extract is analyzed, as the amount of anti-FLAG SPF27 present is saturating for the anti-FLAG antibody. (B) The presence of a FLAG/HA tag does not affect pre-mRNA splicing. Splicing was performed with ³²P-labeled MINX pre-mRNA in HeLa nuclear extract prepared from the control cell line (lanes 1 to 6), FLAG/HA-AD002 cell line (lanes 7 to 12), or FLAG/HA-SPF27 (lanes 13 to 18) for 0 to 90 min as indicated. RNA was recovered and separated on an 8.3 M urea-10% polyacrylamide gel. The ³²P-labeled pre-mRNA and splicing intermediates or products (indicated schematically on the left) were detected by autoradiography.