Figure 4

MBNL and CELF proteins antagonize each other in cells. (A) Co-transfection of HeLa cells with human CELF3 (0.5 μg) and human MBNL1 (1 μg, 2 μg, or 4 μg) protein expression plasmids. Total RNA was isolated from the transfected cells and semi-quantitative RT-PCR was performed using the primers designed in exons 23 and 24 (Figure 1A). The percentage of NF1 exon 23a inclusion is displayed in the bar graph. Error bars indicate standard deviations and n=3. Total protein was collected from the transfected HeLa cells and western blot analysis was performed using anti-Xpress antibody and anti-U1 70K as a loading control. (B) Co-transfection of CA77 cells with HMT-NF1 863/499 reporter (0.5 μg) and human MBNL1 (4 μg) and human CELF3 (1 μg) protein expression plasmids. Total RNA was isolated from the transfected cells and semi-quantitative RT-PCR was performed with the primers designed in exons 1 and 3 of the HMT minigene reporter. The percentage of NF1 exon 23a inclusion is displayed in the bar graph. Error bars indicate standard deviations and n=4. Total protein was isolated from the transfected CA77 cells and a western blot analysis was performed using anti-Xpress antibody and anti-U1 70K as a loading control.