Phosphor p44 42 mapk

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).

After treatment, cells were lysed in NP-40 lysis buffer. Proteins were separated using SDS-PAGE and transferred onto nitrocellulose membrane for antibody detection. The primary antibodies included β-actin from Santa Cruz Biotechnology; AKT and phosphor-AKT from Epitomics; and p44/42 MAPK, phosphor-p44/42 MAPK, estrogen receptor-α, phosphor-estrogen receptor-α and JNK from Cell Signal Technology. In addition, the monoclonal antibody against AGR2, 18A4, was prepared by our laboratory. The secondary antibodies included goat anti mouse and rabbit from Odyssey. Fluorescence was detected with infrared imaging scanner (Odyssey).