Quantification of Dystrophin and Utrophin in mdx Mice

Another cohort of 15-month WBPA-treated (N = 3) and untreated (N = 3) dystrophin-deficient mdx mice were euthanized using CO₂. Ventricular tissues were dissected, minced, and homogenized in modified radioimmunoassay precipitation assay (RIPA) buffer as described before (Altamirano et al., 2014 (link)). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, United States). Proteins were separated on SDS gel, transferred to a nitrocellulose membrane, and incubated with primary. Equal amounts of total protein were separated on 4–12% NuPAGE Novex Bis-Tris SDS-PAGE Gels (Invitrogen Corporation, Carlsbad, CA, United States) and transferred to Immobilon-FL PVDF membrane (Millipore Corporation, Billerica, MA, United States). The following primary antibodies were used; dystrophin and utrophin and then secondary fluorescent antibodies (Abcam, MA, United States). Glyceraldehyde 3-phosphate dehydrogenase (Abcam, Cambridge, MA, United States) was used as protein loading controls. Blots were visualized by Enhanced Chemifluorescence (ECF) (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ, United States) on Storm 860 Imaging System (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ, United States).