Hrp conjugated anti mouse anti rabbit igg

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.