Protein Extraction and Western Blot Analysis

Cells were lysed in RIPA buffer (20 mM Tris, pH 7.3, 150 mM NaCl, 5 mM EDTA, 25 mM NaF, 25 mM sodium pyrophosphate, 1% TritonX, 0.1% SDS, 0.5% deoxycholate, 10% glycerol), sonicated, and centrifuged at 14,000g at 4 °C for 10 min. Protein from aortic tissue was extracted using TRIzol™ Reagent (ThermoFisher) according to Kopec et al.’s^{28 (link)} protocol. Protein concentrations were determined by DC™ protein assay (Bio-Rad). The protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes, which were then incubated with primary antibodies at 4 °C overnight and subsequently with secondary antibodies at room temperature for an hour. Protein bands were detected by Western Lighting Plus-ECL (PerkinElmer) using X-ray films. Antibodies are listed in Supplemental Table 2.