Western blot analysis for AMPA receptor subunits

Rats were deeply anesthetized and perfused with room temperature saline. Spinal cords were harvested and the dorsal half of the lumbar enlargement dissected. Tissue was immediately frozen in extraction lysis buffer (0.5% Triton X-100, 50 mM Tris-HCl, 150 nM NaCl, 1 mM EDTA and 3% SDS). Protein concentrations were determined and Western blot analysis performed. Samples were loaded on a Nu-Page 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA) in MOPS running buffer and transferred onto a single nitrocellulose membrane. Antibodies against the pAKT (ser473, 1:1000; Cell Signaling Technology, Beverly, MA, USA) and GluA1- and GluA2- AMPA receptor subunits were from rabbit (1:1000, Millipore, Temecula, CA, USA). Beta-actin was used as a loading control (Sigma, St. Louis, USA). The membrane was stripped following each analysis. Immunoblots were scanned and a densitometric analysis performed using ImageJ software (U. S. National Institutes of Health, Bethesda, Maryland, USA).