St506 2

Harvested cells were washed with PBS and lysed with Cell lysis buffer for Western and IP (Beyotime, P0013) containing 1 mM phenylmethanesulfonyl fluoride (PMSF) (Beyotime, ST506-2) and centrifuged. Twenty micrograms of protein were separated using SDS-PAGE and transferred to nitrocellulose membranes (Pall, 66485), and then blocked with NcmBlot blocking buffer (NCM Biotech, P30500) for 10 min at room temperature (RT). The membranes were subsequently reacted with primary antibodies at 4°C for 12 h, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. Proteins on the membranes were detected using a SuperSignal West Pico PLUS Chemiluminescent Substrate Kit (ZETA) and visualized via a chemiluminescence apparatus (ImageQuant LAS500.GE).

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).

Protein extracts were isolated from each group of cells with RIPA buffer (P0013, Beyotime) containing 1 mM PMSF (ST506–2, Beyotime). To remove SDS detergent from the samples as well as to reduce and alkylate proteins, the filter-assisted sample preparation method (FASP) was performed subsequently [24 (link)]. Around 200 µg proteins were subjected to digestion following the FASP procedure. The resulting peptides were desalted using the spinnable StageTip protocol followed by nanoLC-MS/MS analysis using an Easynano-LC 1000 HPLC system (ThermoFisher Scientific, San Jose, CA, USA) and a Q-Exactive mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA) [25 (link)]. Protein identification, quantitation, and bioinformatics analyses were searched using MaxQuant and Perseus software respectively.