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. 2010 Jun 2;285(33):25426–25437. doi: 10.1074/jbc.M109.095224

FIGURE 6.

FIGURE 6.

Mbnl1 increases exon 11 incorporation through the downstream intronic enhancer element. A, regulation of IR minigene splicing by Mbnl and CELF4. hIRB minigene was transfected into human HepG2 and HEK293 cells. The rat rIRP minigene was transfected into rat Fao hepatoma cells. Minigenes were cotransfected with the indicated expression vectors for Mbnl1, Mbnl2, Mbnl3, CELF4, or control pCMV vector. Spliced products were analyzed by RT-PCR as before. A representative gel is shown. The percentage of exon 11 inclusion (% IR-B ± S.D., n = 3) is shown below the gel. Statistical analysis was performed by ANOVA. Asterisks indicate statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vector) from three to four independent experiments. B, regulation of rat rIRP splicing regulation in human cell lines by Mbnl1. Rat rIRP minigene was cotransfected into HepG2 and HEK293 cells with or without the Mbnl1 expression vector. A representative gel is shown. The percentage of exon 11 inclusion (% IR-B ± S.D., n = 4) is shown below the gel. Statistical analysis was performed by t test in comparison with the empty pCMV vector. Asterisks indicate statistical significance (*, p < 0.05). C, regulation of human and rat IR minigenes with or without full enhancer element by Mbnl1. Minigenes were cotransfected with Mbnl1 in human and rat cell lines as before. Spliced products were analyzed by RT-PCR. A representative gel is shown. The percentage of exon 11 inclusion (% IR-B ± S.D., n = 3) is shown below the gel. Statistical analysis was performed by t test in comparison with the empty pCMV vector. Asterisks indicate statistical significance (*, p < 0.05). D, knockdown of Mbnl1. hIRB or hIRΔ164 minigenes were cotransfected with 100 nm siRNAs directed against Mbnl1 or scrambled control (Ctrl) siRNA in HepG2 cells. Mbnl1 protein content was measured by immunoblotting with anti-Mbnl1 monoclonal antibodies (upper panels). Blot was stripped and reprobed for β-tubulin protein expression as an internal control. Representative RT-PCR analysis of the exon 11 spliced products from minigene RNA (middle panels) or the endogenous INSR transcript (bottom panel) in Mbnl1 knockdown cells. The percentage of exon 11 inclusion (% IR-B ± S.D., n = 3) is shown. Asterisks indicate statistical significance (*, p < 0.05) versus scrambled siRNA control.