FIGURE 3.

The first RS-like region in NSrp70 is essential for mRNA binding and alternative splicing of CD44 exon v5. A and B, HEK293T cells were cotransfected with GFP_NSrp70 wild-type or mutants and the CD44 minigene. A, the splicing activity and protein expression were determined as described in Fig. 1A. EV, pEGFP-C1_empty vector; Relative in/ex ratio, relative inclusion band/exclusion band ratio; IB, immunoblotting; M, molecular mass. B, after cell lysis, the samples were immunoprecipitated with the indicated antibodies (anti-rabbit IgG or anti-GFP), and then the RNAs were purified from the immunoprecipitated samples for RT-PCR. Whole lysates were used as a positive control for CD44 exon v5. Expression of NSrp70, GFP, and β-actin was confirmed by Western blotting with the indicated antibodies. C and D, the CD44 minigene, GFP_NSrp70 constructs, and Myc_SRSF1 or Myc_SRSF2 were cotransfected into HEK293T cells. Splicing activity and protein expression were determined as described in Fig. 1A. All data are representative of three independent experiments. *, p < 0.05 versus pEGFP-C1_empty vector.