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. 2019 Apr 22;47(11):5922–5935. doi: 10.1093/nar/gkz259

Figure 3.

Figure 3.

Splice site inhibition by downstream complementary sequence prevents usage of alternative splice site. (A–C) Design of the WT/Alt-SS-Open-1, WT/Alt-SS-Open-2, and WT/1928-1930C→U mutants generated from the WT minigene construct. The alternative 5′ SS sequence is highlighted by a blue line. (D) RT-PCR analysis for LMNA/Δ150 expression levels of each mutant. 293T cells were transfected with 1 μg of the indicated plasmids. RNA was harvested at 72 hours post transfection. DsRed expression served as an internal loading control. (E) Quantification of mRNA isoforms expressed as relative usage of the normal vs. alternative 5′ SS. Data are expressed as mean ± s.d. from three independent experiments. UT: untransfected; EV: empty vector; WT: LMNA wild-type minigene construct; MUT: LMNA C1824U mutant minigene construct; NC: negative control (PCR reaction mix without cDNA template).