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Improved immunoblotting methods provide critical insights into phenotypic differences between two murine dysferlinopathy models
Author(s) -
Mueller Amber L.,
Desmond Patrick F.,
Hsia Ruching,
Roche Joseph A.
Publication year - 2014
Publication title -
muscle and nerve
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.025
H-Index - 145
eISSN - 1097-4598
pISSN - 0148-639X
DOI - 10.1002/mus.24220
Subject(s) - dysferlin , endoplasmic reticulum , phenotype , biology , immunoelectron microscopy , desmin , golgi apparatus , unfolded protein response , microbiology and biotechnology , skeletal muscle , immunohistochemistry , endocrinology , immunology , biochemistry , gene , vimentin
ABSTRACT 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. Muscle Nerve 50:286–289, 2014