Figure 2

Nuclear localization and amount of MBNL142–43 isoforms in DM1 myoblasts and muscle tissue. (a) Representative confocal images of DM1 and control primary myoblasts, overexpressing MBNL140–41–42–43 isoforms. Immunofluorescence with specific anti-V5 (MBNL140–41) or -myc (MBNL142–43) tags was performed after 48 h. Scale bar, 10 μm. (b) Representative images of nuclear foci (red) from fixed DM1 muscle, obtained by fluorescence in situ hybridization with a Texas Red labelled (CAG)10 probe. The MBNL1 antibodies (green) anti-P9 and anti-P11 co-localized with foci. Anti-P9 was localized only in the nucleus, in contrast with anti-P11, which was also present in the cytosol. Scale bar, 2 μm. (c) Representative WB analysis of MBNL140–41–42–43 in nuclear and cytosolic fractions from four DM1 patients and four control muscle samples. Proteins, separated in 7.5–17.5% T30C4 SDS-PAGE gels, were blotted onto nitrocellulose and probed with anti-P9 (specific for MBNL142–43 isoforms) and anti-P11 antibodies. Lamin A/C and β-tubulin protein levels were used to normalize the samples from nuclear and cytosolic fractions, respectively. (d) The graph represents the mean±S.D. of at least three experiments. In each lane the ratio between the signal density of MBNL142–43 (upper band) and the sum of the signal density of the two MBNL1 bands (upper and lower) was calculated. The values were expressed as percentage of the value obtained in the control muscle, considered as reference unit. **Significant difference for P<0.01 by Mann–Whitney test