Rabbit anti human nf κb p65 antibody

All group cells were lysed in a buffer containing 50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulphonylfluoride, and 10 µg/mL aprotinin. The extraction and isolation of nuclear and cytoplasmic protein was performed (Wang et al., 2009 (link)) using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Jiangsu, China). The protein concentration was determined by the Bradford assay kit (BioRad Laboratories, Hercules, CA). For this, 25 µg of total protein was separated by SDS-polyacrylamide gel and then transferred to a 0.4 µm PVDF membrane. After blocking with 5% non-fat milk at room temperature for 2 h, the membranes were incubated with rabbit-anti-human NF-κB p65 antibody (human NF-κB had a 90% homology with porcine NF-κB, Santa Cruz Biotechnology, USA, 1:400), rabbit-anti-human IκB-α antibody (human IκB-α had a 97% homology with porcine IκB-α, Santa Cruz Biotechnology, 1:400) and mouse anti-human β-actin (human β-actin had a 99% homology with porcine β-actin, Abcam) for overnight at 4 °C; anti-rabbit and anti-mouse secondary antibodies conjugated to horseradish peroxidase were used at 1:5000 (Santa Cruz Biotechnology). Cross-reactivity was visualised using ECL western blotting detection reagents and then analysed through scanning densitometry by a Tanon Image System.