Depletion of SF2/ASF affects 3′ splice-site usages from exon 1B but not 1A. (A) Schematic of SF2/ASF structural organization and the regions targeted by SF2-shRNAs (sh84, sh584, and sh726). RRM indicates RNA recognition motif. RS, arginine/serine-rich domain. (B) The reduction of endogenous SF2/ASF expression by SF2-shRNAs and the effects on 3′ ss usage. (Top panel) RT-PCR analyses of exon 1A and 1B splicing patterns from RNA isolated from either control nonsilencing shRNA (Non) or SF2-shRNA–treated 1A or 1B minigene expressing MELC. (Middle panels) Southern blot hybridization with exon 2′ (Ex2′) or exon 2 (Ex2) probes. For each shRNA, 4 transductions were performed per experiment. Each experiment was repeated at least 3 times. Mean values ± SD of 3 independent experiments are shown. Standard deviations are omitted in results that consistently have 0% of exon 2′-inclusion product in all 3 reproducible experiments. (Bottom panel) equal amounts of cell lysates from control nonsilencing shRNA (Non)- and SF2-shRNA–treated cells were Western blotted for the presence of SF2/ASF. Expression, SF2/ASF expression levels in shRNA-treated cells relative to nonsilenced cells. β-actin served as loading control.