Improved immunoblotting methods provide critical insights into phenotypic differences between two murine dysferlinopathy models.

INTRODUCTION: We adopted a proteomics-based approach to gain insights into phenotypic differences between A/J and B10.SJL murine dysferlinopathy models. METHODS: We optimized immunoblotting of dysferlin by preparing homogenates of the tibialis anterior (TA) muscle under several different conditions. We compared TA muscles of control, A/J, and B10.SJL mice for levels of dysferlin; dysferlin's partners MG53, annexin-A2, and caveolin-3; and the endoplasmic reticulum (ER) stress marker CHOP. We performed immunoelectron microscopy on control rat TA muscle to determine the precise location of dysferlin. RESULTS: RIPA (radioimmunoprecipitation assay) buffer and sonication improves immunoblotting of dysferlin. The ER stress marker CHOP is elevated in A/J muscle. Dysferlin is localized mostly to membranes close to the Z-disk that have been reported to be part of the Golgi, ER, and sarcoplasmic reticulum (SR) networks. CONCLUSIONS: ER stress might underlie phenotypic differences between A/J and B10.SJL mice and play a role in human dysferlinopathies.