Horseradish peroxidase conjugated goat anti rabbit or anti mouse secondary antibodies

Whole cell lysates from the cells and pulmonary arterioles were prepared in a lysis buffer (Solarbio, China) with protease/phosphatase inhibitor cocktail (Cell Signaling Technology, USA). Then, the concentration of the extracted protein was determined using a BCA protein assay kit (Solarbio, China). After equal amounts of total proteins (20 ug) were separated using SDS-PAGE and then transferred onto a nitrocellulose membrane, the membrane was incubated with primary antibodies against β-actin (8H10D10, CST, USA), HK-II (2106, CST, USA) and proliferating cell nuclear antigen (PCNA) (13110, CST, USA), at a dilution of 1:2500, followed by horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Sigma, USA), at a dilution of 1:2500. Finally, the protein bands were visualized using an enhanced chemiluminescence substrate (Western Lightning VR Plus-ECL), and the relative abundances of the target proteins were measured using ImageQuant TL software. β-actin (CST, 1:2000) served as the internal reference in this study.

The total protein from frozen specimens with the weight about 30–60 mg was extracted using 100–200 μl of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing protease inhibitors (Roche; Basel, Switzerland). Protein concentration was determined using BioTekDc Protein Assay (BioTek; Hercules, CA, USA). Equal amount of lysates (60–90 μg) were subject to SDS-PAGE. The primary anti-DAPK antibody is from LSBio (Life Span BioSciences Inc., Seattle, WA, USA). The horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies were from Sigma-Aldrich (St. Louis, MO, USA).