P ire1

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.