Nf κb p65 polyclonal antibody

RAW 264.7 cells were grown on round glass coverslips (Thermo Scientific, Braunschweig, Germany) in a 6-well plate. They were treated with LPS (1 μg/mL) or LPS plus a single drug (30 μg/mL Ge, 10 μg/mL Pe, 30 μg/mL Ue, 10 μM ga, 20 μM 18ga, 50 μM liq, 4 μM iso, 20 μM pae and 10 μM urs) for 2 h. Dexamethasone (10 μM) was used as a positive control. Then, the cells were washed twice with cold PBS, and fixed with 4% paraformaldehyde for 15–20 min, followed by cold PBS-washing. Then, the cells were blocked with 4% skimmed milk dissolved in PBS for 1 h. After PBS-washing, cells were incubated with NF-κB (p65) polyclonal antibody (1:400) (ThermoFisher Scientific, Fisher Scientific GmbH, Schwerte, Germany) overnight at 4 °C. Followed by a 1 h incubation with Goat anti-Rabbit IgG (H+L) highly cross-adsorbed secondary antibody (1:1000) (ThermoFisher Scientific, Fisher Scientific GmbH, Schwerte, Germany) at room temperature. Afterwards, DAPI was used to stain the cell nucleus at room temperature for 5 min. Then, the coverslips were visualized by fluorescence microscopy (BZ-9000, Keyence, Keyence Deutschland GmbH, Neu-Isenburg, Germany).

The origins of the plant materials (Glycyrrhiza glabra, Paeonia lactiflora, and Eriobotrya japonica) and their extraction processes were previously described [37 (link)]. Lipopolysaccharide (LPS) from Escherichia coli O55:B5, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin, sodium nitrite, sulfanilamide, N-1-napthylethylenediamine dihydrochloride (NED), and dexamethasone were obtained from Sigma-Aldrich (Darmstadt, Germany). NF-κB p65 polyclonal antibody and Goat anti-Rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488, were purchased from ThermoFisher Scientific (Karlsruhe, Germany). Glycyrrhizic acid, 18β glycyrrhetinic acid, isoliquiritigenin, liquiritigenin, paeoniflorin, and ursolic acid came from Baoji Herbest Bio-Tech (Baoji, China).