FIG 7.

Prp18 facilitates NP-RNA complex formation in vitro. (A) Purification of the GST-Prp18 and His-NP proteins. Details are described in Materials and Methods. Purified proteins were loaded onto a 10% SDS-PAGE gel and visualized by Coomassie brilliant blue staining. (B) Gel shift assays were carried out with 5′-³²P-labeled v53 and His-NP. NP-RNA complexes were separated on a 0.6% agarose gel and detected by autoradiography. (C and D) His-NP (lanes 1 to 7, 12.5 fmol) and increasing amounts of GST-Prp18 (lane 2, 0.013 pmol; lane 3, 0.04 pmol; lane 4, 0.13 pmol; lane 5, 0.4 pmol; lane 6, 1.3 pmol; lanes 7 and 8, 4 pmol) were incubated at 30°C for 30 min. After further incubation with 5′-³²P-labeled v53, samples were separated on a nondenaturing gel and subjected to autoradiography. In panel D, the band intensities were quantitatively measured by using ImageJ software, and the averages and standard deviations determined from three independent experiments are shown. The level of significance was determined by Student's t test (*, P < 0.01; **, P < 0.05). AU, arbitrary units.