Effect of SF2/ASF on exon 16 splicing in vivo and in vitro. (A) Effect of transiently expressed SF2/ASF on exon 16 splicing. The WT, EX-3, EX-3.1, and EX-3.2 stably transfected MELCs were transiently transfected with either an empty vector (lanes denoted by -) or a SF2/ASF expression construct (lanes denoted by +). Total RNA was collected 48 hours after transfection, subjected to RT-PCR, and analyzed on a 2% agarose gel. + indicates exon 16 inclusion; and -, exon 16 exclusion. (B) Overexpression of SF2/ASF in EX-3 stably transfected MELCs. The EX-3 stably transfected MELCs were transiently transfected with a SF2/ASF expression construct. Protein was collected at 0, 24, or 48 hours after transfection, fractionated on a 12% PAGE gel, and subjected to immunoblotting analysis with an anti-SF2/ASF antibody. Beta-actin served as a loading control. (C) Purified recombinant SF2/SF2 specifically activates WT exon 16 splicing in an in vitro splicing assay using S100 fraction. In vitro splicing reactions were carried out using HeLa S100 fraction and substrates pre-mRNA derived from either WT or EX-3 minigene constructs in the absence (0 μg) or presence of 0.5 or 1.25 μg of SF2/ASF. Schematic representations of precursors, intermediates, and final products are shown at left. RNA molecular markers in nt are at right.