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. 2014 Feb 19;9(2):e89020. doi: 10.1371/journal.pone.0089020

Figure 5. Splicing of Sgol2 and Tubd1 is responsive to changes in Tra2b concentration.

Figure 5

(A) The murine and human versions of the genomic regions comprising the identified exons of Sgol2 and Tubd1 were cloned into the pSPL3 exon trapping vector. (B) HEK293T cells were co-transfected with the pSPL3 minigene vector and siRNA specific for Tra2b or a TRA2B-GFP expression vector. Western Blot analysis shows efficiently reduced Tra2b protein levels and solid overexpression of TRA2B-GFP. (C) RNA was analyzed for exon inclusion after 48 h by semi-quantitative RT-PCR. (D–G) RT-PCR results were densitometrically quantified. Exon 4 of murine but not human Sgol2 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 43% to 89%. Knock-down of Tra2b is insufficient to reduce Sgol2 exon4 splicing inclusion. Exon 4 of human but not murine Tubd1 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 79% to 94%. Knock-down of Tra2b decreased inclusion of exon 4 from 79% to 71%. Nt, non-treated; scr, scrambled siRNA; si, siRNA against Tra2b; n.s., non significant; error bars show the s.e.m.; significance levels are *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).