Protein Expression Analysis in Endothelial Cells

After treatment, BAECs were lysed in lysis buffer (20 mmol/l Tris–HCl pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l β-glycerophosphate, 1 mmol/l sodium orthovanadate, and 1% Triton X-100, supplemented with protease inhibitor cocktail) for 20 min on ice and then supernatant separated by centrifugation at 12,000 rpm for 10 min at 4°C. Following protein concentration determination by DC protein assay (#5000112, Bio-Rad, Hercules, CA, United States), 25–40 μg of protein were separated in 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by standard Western blotting protocol by probing with antibodies for ACE2 (1:1,000, #ab15348, Abcam, Waltham, MA, United States), TMPRSS2 (1:1,000, #ab92323, Abcam, Waltham, MA, United States), NOX2 (1:250, #611414, BD Biosciences, San Jose, CA, United States), MCP-1 (1:500, #ab9669, Abcam, Waltham, MA, United States), and β-actin (1:1,000, #A2066, MilliporeSigma, St. Louis, MO, United States) as we previously published (23 (link)). The protein bands were visualized by enhanced chemiluminescent methods, and band densities quantified using National Institutes of Health (NIH) Image J program.