Atf6α

Western blot analysis was performed as previously described.²¹ Primary antibodies for ATF6α (Abcam), β‐actin (Abcam), GPX4 (Abcam), PLA2G4A (Santa Cruz Biotechnology), and secondary antibody (Abcam) were applied according to the manufacturer's instructions.

To analyze the levels of UPR‐associated proteins, Western blotting was conducted, as previously described.¹⁷ Briefly, frozen aortae and lungs were crushed to a fine powder in liquid nitrogen using a mortar and pestle, and then dissolved in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Millipore Sigma) at 4°C. The cleared lysates were then suspended in SDS–PAGE sample buffer, separated by 4% to 12% NuPAGE, and blotted onto PVDF membranes. The membranes were then probed with primary antibodies against p‐PERK (Abclonal, #AP0886), p‐IRE1α (Thermo Fisher Scientific, #PA1‐16927), IRE1α (Cell Signaling Technology, #3294), ATF6α (Abcam, ab122897), p‐eIF2α (Cell Signaling Technology, #9721), p‐JNK (Cell Signaling Technology, #9255), CHOP (Cell Signaling Technology, #2895S), BiP (Cell Signaling Technology, #3183S), and cleaved caspase‐3 (Cell Signaling Technology, 9664), followed by incubation with the appropriate horseradish peroxidase–conjugated secondary antibodies (1: 5000 dilution). Membranes were developed with a chemiluminescent substrate and imaged using the KwikQuant Imager system (Kindle Biosciences, LLC). Target protein band densities were quantified and normalized to those of GAPDH, beta‐actin, or beta‐tubulin, as indicated. Band intensity data are expressed relative to the control group (Young/ND).