SDS-PAGE Protein Extraction and Analysis

SDS-PAGE was performed as described previously [12 (link)]. In brief, cells were washed twice with ice-cold DPBS and lysed in RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with 1 × protease inhibitors cocktail (Sigma, St Louis, MO, USA) and 1 × phosphatase inhibitors cocktail (Roche Applied Science, Hercules, CA, USA). The samples were then centrifuged for 5 min at 10,000× g. Protein concentration was determined by BCA (bicinchoninic acid assay) protein assay (Thermo Fisher, Waltham, MA, USA). 20 μg of protein was denatured using Laemmli SDS buffer, heated to 95 °C for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis. ECs were pooled together from ≥10 murine aortas for each group/lane and samples were loaded in three wells. When proteins were at a similar molecular weight, parallel blots were employed to overcome the ineffectiveness of the restriping and reprobing process.