Endothelial Cell Protein Expression Analysis

HUVECs and lung endothelial cells were lysed with radio-immunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Danvers, MA, USA), sonicated, and centrifuged. The supernatants were harvested, and the protein concentration was measured using a bicinchoninic acid (BCA) kit (Thermo-Fisher Scientific); 20 μg of the lysate was analyzed as described previously [8 (link)]. The following primary antibodies were purchased: p16, p21, p62, and Sirt3 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); p53, p-RB, t-RB, PINK1, Parkin, Myc, and IDH2 antibodies from Abcam (Cambridge, UK); LC3 antibody from NOVUS Biologicals (Centennial, CO, USA); COX IV antibody from cell signaling (4844s); and p62 and β-actin antibodies from Sigma-Aldrich. Mouse and rabbit secondary antibodies were obtained from Thermo-Fisher Scientific (Waltham, MA, USA).