Western Blot Analysis of Signaling Proteins

Western blots were performed as previously described [25 (link),26 (link)]. In brief, heart tissue samples were homogenized in ice-cold lysis buffer and protein concentration was determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Heart homogenate proteins were then resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. For reprobing, membranes were stripped with Restore Western Blot Stripping Buffer from Thermo Scientific (Rockford, IL, USA). Rabbit polyclonal antibodies against AMPK, phosphor-AMPK (Thr¹⁷²), ACC, phosphor-ACC (Ser⁷⁹), p44/42 MAPK, phosphor-p44/42 MAPK (Thr²⁰²/Tyr²⁰⁴), p38 MAPK, phosphor-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²), SAPK/JNK, phosphor-SAPK/JNK (Thr¹⁸³/Tyr¹⁸⁵) and horseradish peroxidase-linked secondary antibody were purchased from Cell Signaling (Danvers, MA, USA).