Sc 7351

NRVMs were washed with cold PBS and lysed with 1
× SDS-PAGE sample buffer (45 mM Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, 0.01% bromophenol blue, and 0.05 mM DTT) supplemented with 1 mM PMSF and a protease inhibitor cocktail (Roche, Mannheim, Germany). The lysates were boiled at 99°C for 10 min, separated by 12% SDS-PAGE, and then transferred onto PVDF membranes. The PVDF membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at a room temperature, followed by overnight incubation at 4°C with the appropriate primary antibodies against C/EBP homologous protein (CHOP) (1:500, sc-7351; Santa Cruz Biotechnology, Dallas, USA), ubiquitin (1:500, sc-8017; Santa Cruz Biotechnology), GRP78 (1:500, BS6479; Bioworld Technology, Shanghai, China), eukaryotic initiation factor 2α (eIF2α) (1:500, BS3651; Bioworld Technology), and phosphorylated eIF2α at Ser51 (p-eIF2α) (1:500, BS4787; Bioworld Technology). The membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (1:10,000, BS13278, BS12478; Bioworld Technology). Finally, the protein blots were visualized using an enhanced chemiluminescence kit (Bio-Rad, Hercules, USA) with the Fluor Chem E System (Cell Biosciences, Santa Clara, USA). To promote ER stress in NRVMs, we used thapsigargin (1.0 μM, T9033; Sigma-Aldrich Chemical) as a positive control.