Peroxidase conjugated secondary antibody

Heart tissues from infarct areas were collected, and then crushed by a tissue homogenizer. Cultured cells were washed with ice-cold PBS, and collected by a cell scraper. Both the tissues and cells were lysed by ice-cold RIPA buffer supplemented with 1% PMSF for 30 min, then centrifuged at 12,000 rpm for 10 min at 4 °C. The protein concentrations were detected by means of the BCA method. Equal amounts of proteins were loaded onto SDS-PAGE for electrophoresis and subsequently transferred onto the activated PVDF membranes. The membranes were blocked with 5% BSA and probed with primary antibodies against GPX4, ACSL4, FTL, BCAT1, BCAT2, Keap1, Nrf2, HO-1, GAPDH, and β-actin (dilution 1:1000, 1:500, 1:1000, 1:1000, 1:500, 1:2000, 1:1000, 1:1000, 1:8000, 1:10,000) overnight at 4 °C. Membranes were then probed with peroxidase-conjugated secondary antibody at 1:10,000 dilution (Bioworld Technology, Louis Park, MN, USA). The protein signal was detected using the ECL plus system (Amersham, Arlington Heights, IL, USA) and the band densities were determined by Bio-Rad Laboratories.

The proteins from small intestines for western blotting analysis were obtained as previously described [30 (link)]. The primary antibodies against ZO-1 (1 : 1000, Abcam, USA), VE-cadherin (1 : 1000, Santa, USA), occludin (1 : 200, Abcam, USA), ROCK-1 (1 : 500, Santa, USA), MLC (1 : 1000, CST, USA), phospho-MLC (1 : 1000, CST, USA), and GADPH (1 : 2000, Bioworld, USA) were used, followed by incubation with peroxidase-conjugated secondary antibodies (1 : 8000, Bioworld, Louis Park, USA). A BCA kit was used to quantify protein and add loading buffer to obtain the western blotting sample, and the loading volume is 30 μg. Bands were demonstrated by enhanced chemiluminescence (ECL).

Total cell lysates were solubilized in ice-cold RIPA lysis buffer (Beyotime Biotechnology, P0013B, China) consisting of protease and phosphatase inhibitor cocktails (HY-K0010, HY-K0021, HY-K0022, MCE, United States). The membranes were blocked with 5% bovine albumin for 2 h at room temperature prior to incubation with the indicated primary antibodies. The signals were detected with the following antibodies following standard procedures: phosphorylated STAT3 (CST, #9139), Atrogin-1 (Satan Cruz, sc166806), MuRF-1 (Santa Cruz, sc398608), MHC (R&D Systems, #MAB4470), p62 (CST, #5114s), LC3 (Proteintech, 14600-1-AP), phospho-P70 S6K (Beyotime, AF5899), phospho-EIF4EBP1 (Beyotime, AF5806), phospho-mTOR (Beyotime, AF5869), GDF8/Myostatin (Proteintech, 19142-1-AP), iNOS (Affinity, AF0199), phospho-Akt (Ser473) (CST, #4060), phospho-AMPK (Abcam, ab133448), ATGL (CST, #2439), HSL (CST, #18381), UCP-1 (Satan Cruz, sc518171) and GAPDH (Proteintech, 60004-1-Ig). Subsequently, the membranes were washed and incubated for 2 h at room temperature with peroxidase-conjugated secondary antibodies (Bioworld). Following several washes, chemiluminescent images of immunostained bands on the membranes were recorded on X-ray films using the enhanced chemiluminescence (ECL, FUDE, FD8020, China) system according to the manufacturer’s instructions.

Total cell lysates were solubilized in ice-cold RIPA lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitor cocktails (MedChemExpress (Monmouth Junction, NJ, USA). Incubation with the primary antibodies was performed after blocking the membranes with 5% bovine albumin at room temperature for 2 h. Standard procedures were followed for the detection of the signals using the following antibodies: STAT3 (CST, #9145), phosphorylated STAT3 (CST, #9139), atrogin-1 (Satan Cruz, sc166806), MuRF-1 (Santa Cruz, sc398608), MHC (R&D Systems, #MAB4470), p62 (CST, #5114s), LC3 (Proteintech, 14600-1-AP), cleaved caspase3 (CST, #9664T), cleaved PARP (CST, #5625), phospho-P70 S6K (Beyotime, AF5899), phospho-EIF4EBP1 (Beyotime, AF5806), GLUT1/SLC2A1 rabbit mAb (Abclonal, A11727), Bdh1 rabbit pAb (Abclonal, A3763), HMGCS2 antibody (Affinity, DF14319), and GAPDH (Proteintech, 60004-1-Ig). Subsequently, at room temperature, the membranes were washed and incubated for 2 h with peroxidase-conjugated secondary antibodies (Bioworld). The enhanced chemiluminescence (ECL) system was used to record chemiluminescent images of immunostained bands on membranes on X-ray films, following the manufacturer’s protocol.

Cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Beyotime, China) to extract total proteins. After quantification, 25 μg of protein was loaded and separated with 4–12% Bis-Tris SurePAGE gels (GenScript, China) and then transferred onto PVDF membranes (Merck Millipore, Germany). After blocking with 5% BSA for 1.5 h, the membranes were incubated overnight at 4°C with anti-Sox9 (1:1000, Abcam, UK), anti-Sox2 (1:1000, Proteintech, USA), anti-Klf4 (1:1000, SAB, USA), anti-cMyc (1:1000, Novus, USA), anti-TLR4 (1:1000, Proteintech, USA), anti-YAP1 (1:1000, CST, USA), anti-CTGF (1:1000, Abcam, UK), and anti-β-actin (1:4000, Bioworld, USA) antibodies. After incubation with peroxidase-conjugated secondary antibodies (1:5000, Bioworld, USA) for 1.5 h at room temperature, the membranes were visualized using enhanced chemiluminescence detection reagents (GE Healthcare, USA).