Ab62484

HUVECs were lyzed using RIPA lysis buffer (Beyotime, Shanghai, China) and cell supernatants were collected by centrifugation. The protein concentration was determined using BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were isolated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA, membranes were incubated with primary antibodies against Bcl-2 (Abcam, ab32124, 1:1000), Bax (Abcam, ab243140, 1:500), Cleaved caspase-3 (Abcam, ab2302, 1:500), Caspase-3 (Abcam, ab184787, 1:2000), Beclin-1 (Abcam, ab207612, 1:2000), Atg5 (Abcam, ab108327, 1:10,000), p62 (Abcam, ab207305, 1:1000), GRP78 (Abcam, ab108615, 1:10,000), CHOP (Abcam, ab11419, 1:1000), Caspase-12 (Abcam, ab62484, 1:2000), XBP-1 (Abcam, ab220783, 1:1000) and GAPDH (Abcam, ab32124, 1:1000) overnight at 4°C. Membranes were washed in TBST, followed by incubation with secondary antibodies (Abcam, ab205718, 1:50,000; Abcam, ab205719, 1:20,000) for 1 h at room temperature. Protein bots were visualized using an enhanced chemiluminescence (ECL) detection kit (Beyotime, Shanghai, China).

Total proteins were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and the concentration of the protein samples was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime). Proteins were subjected to SDS-PAGE and then electrophoretically transferred onto PVDF membranes. Membranes were incubated with primary antibodies overnight at 4°C after blocking in 5% nonfat milk in TBST buffer, which was then coincubated with horseradish peroxidase (HRP)-conjugated anti-mouse/anti-rabbit IgG (Thermo Fisher Scientific). Proteins were visualized using a detection system of enhanced chemiluminescence (ECL), and the bands were analyzed using BandScan ImageJ software. The primary antibodies used in this study were as follows: anti-CHOP (diluted 1 : 1000, ab11419, Abcam), anti-caspase 12 (diluted 1 : 1,000, ab62484, Abcam), anti-GRP78 (diluted 1 : 100, ab21685, Abcam), anti-NLRP3 (diluted 1 : 500, ab214185, Abcam), anti-ASC (diluted 1 : 1000, ab155970, Abcam), anti-pro-IL-1β (diluted 1 : 500, ab2105, Abcam), anti-IL-1β (diluted 1 : 1500, ab9722, Abcam), anti-p-NF-κB (p-p65) (diluted 1 : 2000, ab86299, Abcam), anti-NF-kB p65 (diluted 1 : 300, ab19870, Abcam), anti-TLR4 (diluted 1 : 500, ab13556, Abcam), anti-MyD88 (diluted 1 : 1000, ab133739, Abcam), and anti-β-actin (diluted 1 : 1000, ab8226, Abcam). β-Actin was used as an internal control.