Protein Extraction and Western Blot Analysis

Total proteins were isolated from the cardiac and pulmonary tissues using a radioimmunoprecipitation assay buffer as described previously [28 (link)]. The protein concentration was measured using the bicinchoninic protein assay kit. Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to a polyvinylidene difluoride membrane (pore size: 0.45 μm; Merk Millipore, MA, USA). The membrane was blocked with 5% skim milk in Tris-buffered saline containing Tween-20 (TBST) (20 mM Tris, 200 mM NaCl, and 0.04% Tween 20) for 1 h at 25°C and probed with the primary antibodies (1 : 1000) overnight at 4°C. Next, the membrane was washed thrice with TBST for 5 min and incubated with the anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (1 : 3000) for 1 h at 25°C. Immunoreactive signals were detected using Immobilon western blotting detection reagents (EMD Millipore, Billerica, MA, USA). The intensities of the protein bands were quantified using ImageJ software (https://imagej.net/).