Effects of Tip110 expression on YB-1-mediated alternative splicing of the CD44 minigene. A. Schematic of the CD44 minigene containing the variable exon 5 (V5). The locations of the primer set used for RT-PCR were marked by arrows, the dotted lines indicated two alternative splicing possibilities: one with V5 (top, +V5), the other without V5 (bottom, -V5). B. 293T cells were transfected with the CD44 minigene plasmid, pYB-1-Myc, pTip110-HA, or both. Input amounts of plasmids were shown at the top, all were in micrograms (μg). Total RNA was isolated and used for RT-PCR. The ratio of V5 inclusion (+V5/–V5) was determined by densitometric scanning and the ImageJ software and was normalized to that transfected with the CD44 minigene only. Expression of YB-1 and Tip110 was determined by Western blotting. β-actin was the loading control. C. 293T cells were transfected with the CD44 minigene, pYB-1-Myc, pTip110-HA, or each of the Tip110 mutants. The V5 inclusion and quantitation were performed as stated above.