Western Blot Analysis of Signaling Proteins

Western blot was performed using fresh or frozen tissue, as previously described [11 (link)]. The following antibodies were used; phospho-Thr¹⁷²-AMPKα (Cell Signaling Technology Cat# 2535S RRID:AB_331250), AMPKα (Cell Signaling Technology Cat# 2532S RRID:AB_10694064), phospho-Ser⁷⁹-ACC Cell Signaling Technology Cat# 3661S RRID:AB_330337) and phospho-Ser⁴²⁸-LKB1 (Cell Signaling Technology Cat# 3482S RRID:AB_2198321), nNOS (Abcam Cat# ab1376 RRID:AB_300614), phospho nNOS Ser¹⁴¹⁷, equivalent to human Ser¹⁴¹² (Abcam Cat# ab90443 RRID:AB_2049208) and β-actin (Sigma-Aldrich Cat# A3853 RRID:AB_262137), followed by horseradish peroxidase-conjugated, goat anti-rabbit secondary antibody (Jackson ImmunoResearch Cat# 111-035-045 RRID:AB_2337938). Protein concentrations were determined using protein assay dye from Bio-Rad Laboratories (Hercules, CA). Densitometry of protein expression was performed using a GS800 calibrated densitometer from Bio-Rad laboratories (Hercules, CA).