Western Blot Analysis of Muscle Atrophy

For Western blotting, total protein was isolated from mouse quadriceps or adipose tissue by homogenization in RIPA buffer with complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche, Pleasanton, CA)[33 (link)]. Protein quantification was determined by the Bradford protein assay, and samples were equally loaded on 10% polyacrylamide gels (NuPage, Grand Island, NY), electrophoresed at 200V, electrotransferred to PVDF membranes, and probed with antibodies to Atrogin-1 (1:500; ECM Biosciences, Versailles, KY), Myostatin (1:200; Abcam, Cambridge, MA), MuRF1, ZAG (1:500; both Santa Cruz Biotechnology, Dallas, TX), p21 (1:200; Santa Cruz), β-actin, Smad2, p-Smad2/3 (1:1000; all Cell Signaling, Danvers, MA), TGF-β (1:1000; Abcam), and Ezrin (1:1000; BD Transduction Laboratories, San Jose, CA). Quantification was performed by measuring integrated density and normalizing to loading controls. For immunohistochemical analysis, paraffin-embedded tissue sections were stained using polyclonal anti-TGF-β (1:100; Abcam, Cambridge, MA), anti-p21 (1:100, Santa Cruz), or anti p-Smad 2/3 (1:50, Santa Cruz) and the corresponding isotype controls[33 (link)].