Nf κb p65 rabbit polyclonal antibody

Nuclear and cytoplasmic lysates containing equal amounts of protein (25 μg) were equally loaded on 12% SDS–polyacrylamide gel and transferred to PVDF membranes (Millipore) using a Mini-Protean 2 electrophoresis system (Bio-Rad Laboratories). After blocking with 5% skimmed milk in TBS plus 0.1% Tween-20, the membranes were incubated with NF-κB p65 rabbit polyclonal antibody (Proteintech) overnight at 4 °C followed by HRP-conjugated goat anti-rabbit IgG (Trans Gen Biotech). Detection was visualized using an ECL assay kit (Thermo Fisher Scientific, Inc.). Images were subsequently analyzed using Image J software to quantify the protein expression (National Institutes of Health, USA). All blots were repeated in triplicate.

The cells were seeded on coverslips in 12-well plates at a density of 15,000 cells/cm.² Pre-treatment with or without catalpol (500 μM) or TAK-242 (1 μM), cells were incubated with Ang II (0.1 μM) or LPS (1 μg/mL) for 24 h. After being fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.3% Triton X-100 for 30 min at room temperature, cells were incubated in immunol staining blocking buffer (Beyotime, Beijing, China) for 1 h. Primary antibody was used for overnight incubation: NF-κB p65 rabbit polyclonal antibody (1:500, Proteintech, Chicago, IL). After that, cells were incubated with fluorescent-labelled secondary antibody (1:1000, Proteintech, Chicago, IL) for 1 h in the dark at room temperature. 4′,6-diamidino-2-phenylindole (DAPI) was used to staining the nuclei for 10 min. Images were taken with a fluorescence microscope (DS-Qi2, NIKON, Tokyo, Japan) and semi-quantified by Image J.