Fig. 8.

Expression of the Dp71 isoform in LRMD muscles. a Comparative immunohistochemistry analysis of biopsies from the biceps femoris of a healthy dog, a GRMD dog and a LRMD dog. Three antibodies were used: MANEX1A (N-terminal part), Dys1 (Rod domain, downstream of the mutation), Dys2 (C-terminal part). As expected, the staining was positive in the muscle of the healthy dog, and negative for the GRMD dogs regardless of the antibody used. The sample obtained from the LRMD dog was negative for MANEX1A and Dys1, and positive for Dys2, but the staining was found to be heterogeneous among fibres. b Western blotting using the Dys2 antibody (C-terminal part). Muscle protein extracts from two healthy animals were loaded in wells 1 and 4 (50 μg); a normal size band corresponding to the full-length dystrophin (427 kD) was seen in these samples. Muscle protein extracts from LRMD muscles (250 μg) were loaded in wells 2 (LRMD3, biceps femoris) and 3 (LRMD3, interosseus muscle); a band at around 70 kD was observed in these lanes indicating that the protein detected was truncated. c RT-PCR using a forward primer designed to bind at the junction between the specific first exon of the Dp71 and the exon 64, and a reverse primer at the junction between exons 65 and 66. cDNA from a canine liver was used as a positive control showing a band of the expected size (164 bp). A very faint band was observed using the cDNA from a muscle sample obtained from a healthy dog and no band was observed for the GRMD dog. Conversely, in the three samples obtained from LRMD muscles (LRMD8, sartorius cranialis, biceps femoris, tibialis cranialis) a band at the same size as the positive control, though less pronounced, was seen, indicating that the Dp71 transcript is present in LRMD muscle. Sart sartorius cranialis muscle, BF biceps femoris muscle, TC tibialis cranialis muscle