P perk

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).

Western blotting was performed as previously described [34 (link)]. In brief, skeletal muscle and treated cells were lysed in ice-cold RIPA buffer with PMSF protease inhibitor (Beyotime). The supernatant was obtained by centrifugation, and the concentration of protein was determined by a BCA assay kit (Beyotime). Equal amounts of proteins from different groups were loaded and separated on 6–15% SDS–PAGE gels, transferred onto nitrocellulose membranes (Pall Corporation) and blocked for 2 h at 37 °C with 5% milk-TBST. Membranes were incubated either overnight at 4 °C with primary antibodies against: SelK (Abcam, 1: 500), MyoD (Abclonal, 1:1000), MyoG (Abclonal, 1:1000), MyHC (Abclonal, 1:1000), GRP78 (Abclonal, 1:1000), ATF6 (Abclonal, 1:1000), phospho-IRE1 (p-IRE1, Abclonal, 1:500), phospho-PERK (p-PERK, Abclonal, 1:500), phospho-eIF-2α (p-eIF-2α, Abclonal, 1:1000), CHOP (Abclonal, 1:1000), LC3B (Abclonal, 1:1000), p62 (Abclonal, 1:1000), cleaved Caspase3 (cle-Cas3, Abclonal, 1:1000), Bcl2, Bax (My lab, 1:400) and GAPDH (Servicebio, 1:1000). Then, the membranes were washed with TBST three times and incubated with HRP-conjugated secondary antibodies (ImnunoWay, 1: 8000) at temperature for 1 h. The bands were visualized by using an ECL kit (Kangweishiji Biotechnology) and an Azure imaging Biosystem C300.