Utrophin Expression in mdx Muscles

Western blots were performed on whole muscle lysates as previously described [26 (link)]. Briefly, the gastrocnemius muscles treated mdx^4cv muscles and contralateral controls (n = 4) were ground in liquid N₂ and homogenized in extract buffer (50 mM Tris-HCl, 150 mM NaCl, 0.2% SDS, 24 mM Na deoxycholate, 1% NP40, 47.6 mM Na Fluoride, 200 mM Na Orthovanadate, Roche, Basel, CH). Protein concentration of whole muscle was determined by Coomassie Plus Bradford Assay (Thermoscientific, Rockford, IL). Equal amounts of protein (20 μg) were resolved on a 4–12% SDS polyacrylamide gel. The blots were incubated in N-terminal anti-utrophin (1:1000; kind gift from Stanley C. Froehner) overnight at 4°C. The GAPDH antibody (1:50,000; Sigma, St. Louis, MO) was used as a loading control as its expression was unchanged when comparing the treated and untreated mdx^4cv muscles. The primary antibodies were detected with IgG HRP secondary antibodies (1:25,000; Jackson ImmunoResearch Labs). The blots were developed with ECL plus (Thermoscientific, Rockford, IL) and scanned with the Storm 860 imaging system (GE Healthcare Lifesciences, Piscataway, NJ). The band intensity was measured using Image J software (NIH).