FIG. 7.

RNA electrophoretic mobility shift analysis of mutated PCBP2 proteins with stem-loop I and stem-loop IV RNA sequences. Reaction mixtures contained a final RNA probe concentration of 0.1 nM and were resolved on native 4% polyacrylamide-5% glycerol-0.5× TBE gels at 4°C. (A) [α-³²P]UTP-labeled stem-loop I RNA was incubated with increasing molar amounts of mutated PCBP2 proteins including mKH1-2 (lanes 2 to 7), mKH2-6 (lanes 8 to 13), mKH3-4 (lanes 14 to 19), or mKH3-3 (lanes 20 to 25). (B) [α-³²P]UTP-labeled stem-loop IV RNA was incubated with increasing molar amounts of mutated PCBP2 proteins including mKH1-2 (lanes 2 to 7), mKH2-6 (lanes 8 to 13), mKH3-4 (lanes 14 to 19), or mKH3-3 (lanes 20 to 23). Lane 1 of both panels represents RNA probe incubated in buffer alone. The mobilities of the free probe are indicated for both panels. (C) Three micrograms of wt rPCBP2 (lane 1) and each of the mutated PCBP2 proteins, mKH1-2 (lane 2), mKH2-6 (lane 3), mKH3-3 (lane 4), and mKH3-4 (lane 5), that were used in these and the following experiments were analyzed by electrophoresis on 12.5% polyacrylamide gels containing SDS and visualized by Coomassie blue staining. For molecular mass comparisons, the molecular masses (in kilodaltons) and relative mobilities of a set of protein standards are indicated to the right of the panel.