title: Improved immunoblotting methods provide critical insights into phenotypic differences between two murine dysferlinopathy models.
abstract: BACKGROUND
  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.
authors.affiliation: University of Maryland School of Medicine, Department of Physiology, Baltimore, Maryland, 21201, USA.
authors.forename: Amber L
authors.initials: AL
authors.lastname: Mueller
authors.forename: Patrick F
authors.initials: PF
authors.lastname: Desmond
authors.forename: Ru-Ching
authors.initials: RC
authors.lastname: Hsia
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Roche
mesh_terms: D000818:Animals; D017306:Annexin A2 / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D051244:Caveolin 3 / Q000378:metabolism; D004195:Disease Models, Animal; D059865:Endoplasmic Reticulum Stress / Q000502:physiology; D015151:Immunoblotting; D008565:Membrane Proteins; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology; D049288:Muscular Dystrophies, Limb-Girdle / Q000175:diagnosis* / Q000503:physiopathology*; D010641:Phenotype; D015531:Radioimmunoprecipitation Assay; D013045:Species Specificity; D051742:Transcription Factor CHOP / Q000378:metabolism
chemical_list: D017306:Annexin A2; D002352:Carrier Proteins; D051244:Caveolin 3; C497250:Ddit3 protein, mouse; C538824:MG53 protein, mouse; D008565:Membrane Proteins; D051742:Transcription Factor CHOP
keywords: ER stress; dysferlin; dysferlin binding proteins; immunoblotting; skeletal muscle