Quantifying Cardiac Inflammatory Proteins

Heart proteins were extracted using RIPA lysis buffer. Total protein was quantified by Bradford assay and the expression of specific proteins was determined by Western blotting as described previously (Chavali et al., 2014 (link)). The primary antibodies for TNF-α (Cell Signaling, 3707s), IL-10 (EMD Millipore, ABF13), and β-actin (Santacruz Biotech, sc-47778) were diluted 1:1000 and blots were incubated in primary antibody overnight at 4°C. HRP-linked secondary antibodies against rabbit (Santacruz Biotech, sc-2054) or mouse (Santacruz Biotech, sc-2005) were used in 1:4000 dilution. Blots were incubated in secondary antibodies for 1 h at room temperature (RT). The membranes were exposed with HRP substrate and imaged with BioRad ChemiDoc. Densitometry analyses was performed by ChemiDoc software (BioRad, CA, USA).