FIGURE 6.

β-Exon splicing pattern of Mef2D minigene constructs in C2C12 cells. A, schematic diagrams of WT and mutant (ΔT5) Mef2D minigene constructs consisting of exons 6, β, and 7 and the intervening introns are shown. B, WT Mef2D minigene construct was cotransfected into C2C12 cells with either pcDNA3.0FLAG vector (−), an expression plasmid for the ZFmt, or the WT-MBNL3 expression plasmid (WT). Approximately 24 h post-transfection, total RNA was isolated, and the β-exon splicing pattern was determined by RT-PCR. Amplified products were loaded onto native polyacrylamide gels and detected by ethidium bromide staining. The intensity of (+)β and (−)β splice products was determined using National Institutes of Health Image J software and used to calculate the percentage of total Mef2D transcript for each isoform. Percentages from three independent experiments (n = 5) are provided. C, level of WT and ZFmt MBNL3 protein expressed from the transfected expression vector was determined by Western blotting. Tubulin protein levels were measured to control for variations in protein load. D, WT or ΔT5 Mef2D minigene was transfected into C2C12 cells in the absence (−) or presence (+) of WT-MBNL3 expression plasmid. The samples and resulting data were analyzed as described in B. E, expression levels of MBNL3 protein were determined by Western blotting. Tubulin protein levels were used to monitor for differences in protein loading. Statistically significant differences are indicated by the asterisks, and the p values are provided.