Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain
Figure 4
SC35 and ASF/SF2 are negative regulators of CASK E24a. (a) Schematic illustrates the copy number of ASF/SF2 and SC35 motifs in each exon ± ~250 bases of flanking intron. The CASK_E24a splicing reporter is shown below with exons represented as boxes and introns as lines. E24a is inserted as the middle exon with flanking intron sequences (thick grey lines); nucleotide lengths for each segment are indicated. Open arrows indicate primer positions in exons 1 and 3 that were used for the amplification of exon included and skipped products. (b) The CASK_E24a splicing reporter was transfected into MEF cells engineered with an inducible HA-tagged SC35, or ASF/SF2 expression cassette. Gel panels show splicing patterns after cells were cultured in medium containing Dox, which leads to depletion of SC35 (lanes 4–6) or ASF/SF2 (lanes 10–12). Control cells retain SC35 (lanes 1–3) or ASF/SF2 (lanes 7–9) expression when cultured without Dox. Each lane represents an individual transfection experiment. Graph illustrates the average % exon inclusion values for the gel panels shown. Numbers at bottom of graph indicate net change in splicing pattern (c) Depletion of SC35 and ASF/SF2 were verified by Western blotting with an antibody specific for the HA tag. β-actin, loading control.
