Fig. 5. Binding of SPF45 competes out U2AF⁶⁵ on truncated PPTs in short introns.

a Affinity pull-down experiments of biotinylated RNA indicates U2AF⁶⁵ binding to the short intron only if SPF45 was depleted. Biotinylated pre-mRNAs including short HNRNPH1 intron (56 nt) and control AdML intron (231 nt) were incubated with nuclear extracts from either control siRNA- or SPF45 siRNA-treated HEK293 cells. The biotinylated RNA-bound proteins were pulled down with streptavidin-coated beads and analyzed by Western blotting with antibodies against SF3b155, SPF45, and U2AF⁶⁵. b The graph shows quantification of the bands on Western blots (panel a as the representative blots). SPF45 and U2AF⁶⁵ scanned data were normalized to SF3B155 (SPF45/SF3B155 and U2AF⁶⁵/SF3B155) and plotted. Means ± SEM are given for four independent experiments and two-tailed Student t-test values were calculated (Left panel: p = 0.2982 for AdML vs HNRNPH1 intron in Control NE; p = 0.6973 for AdML vs HNRNPH1 intron in SPF45-KD NE; Right panel: p = 0.0142 for AdML vs HNRNPH1 intron in Control NE, p = 0.4647 for AdML vs HNRNPH1 intron in SPF45-KD NE). *P < 0.05, ^n.s. P > 0.05. Source data of all the above panels are provided as a Source Data file.