Protein Expression Analysis in Myocardial Infarction

Rats of each group were sacrificed when the 4-week treatments were finished, and hearts were snap-frozen and stored at −80°C. Myocardial tissues from the infarct border zone (2 mm away from the infarct edge) (Kohno et al., 2008 (link)) were sheared and homogenized in ice-cold lysis buffer system containing RIPA lysate (1 mL, 89900, ThermoFisher, China), protease inhibitors (10 μL, ST506-2, Beyotime, China), and phosphatase inhibitors (10 μL, P1260, Applygen, China). Subsequent steps including protein sample extraction, concentration normalization, and detection of specific protein were conducted according to our previous description (Wu et al., 2017 (link)). The details of primary antibodies are listed below: p-AMPK (Thr172; 40H9, CST, United States), AMPK (D5A2, CST, United States), Cx43 (3512, CST, United States), TGF-β (ab92486, Abcam, China), Collagen-I (14695-1-AP, Proteintech, China), Collagen-III (22734-1-AP, Proteintech, China), IL-1β (ab9722, Abcam, China), IL-6 (ab9324, Abcam, China), TNF-α (ab220210, Abcam, China), TLR-4 (ab22048, Abcam, China), MyD88 (ab2064, Abcam, China), p-P65 (Ser536; ab86299, Abcam, China), and P65 (ab16502, Abcam, China), GAPDH (ab181602, Abcam, China).