Evaluation of antibody reactivity to dystrophin products: does Dp40 really exist?

Duchenne and Becker muscular dystrophies (DMD/BMD) are caused by mutations in the dystrophin gene. The intron-62 promoter produces Dp71 and the putative shorter isoform Dp40, but antibody specificity and the existence of Dp40 protein remain controversial. Here, we systematically evaluated the reactivity of commonly used anti-dystrophin antibodies toward Dp71 isoforms and Dp40. The Abcam DMD and Novocastra/Leica DYS2 antibodies were Dp71d-specific, whereas the Proteintech DMD, 7A10, and N-terminal Dp71/40 antibodies recognized both Dp71d and Dp71f isoforms. Importantly, ectopic Dp40 was detected using only N-terminal Dp71/40 antibody, and signals previously attributed to Dp40 likely represent proteolytic fragments of Dp71. Sequence analysis further indicates that the Dp40 transcript does not appear to contain a functional polyadenylation signal within its intron 70-derived sequence and instead retains downstream exon-intron sequences, suggesting that it may represent an atypical transcript. These findings define antibody specificity and raise questions regarding the existence of Dp40 protein, providing a framework for future studies on Dp71 and Dp40.