Phospho nf κb antibody

The fruiting bodies of the wild mushroom A. hemibapha subspecies javanica were purchased from the local market in Chiang Mai, Thailand, in 2018. They were carefully washed with tap water, and dried using a hot air oven at 60 °C for 3 h. The dried sample was finely milled with a grinder into powder, and stored at −20 °C. RPMI-1640 cell culture medium, fetal bovine serum (FBS), streptomycin, and penicillin were purchased from Gibco Life Technologies (Grand Island, NY, USA). Griess reagent was purchased from Sigma-Aldrich (NSW, Australia). The EZ-Cytox cell viability assay kit (WST-1) was purchased from Daeil Lab Service Co., Ltd., Korea. The anti-complement receptor 3 antibody (anti-CR3), and anti-toll-like receptor 4 antibody (anti-TLR4) were obtained from Abcam (Cambridge, MA, USA). The phospho-NF-κB antibody, phospho-p38 antibody (MAPK), phospho-ERK (MAPK) antibody, and phospho-JNK antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals, and reagents used in this study were of high analytical grade.

Liver protein samples were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto Immun-Blot PVDF membranes (Bio-Rad Laboratories, K.K., Tokyo, Japan). The membranes were then incubated overnight at 4 °C with IL-1β antibody (#12242; Cell Signaling,), TNF-α antibody (#11948; Cell Signaling), NF-κB antibody (#8242; Cell Signaling), phospho-NF-κB antibody (#3033; Cell Signaling) and anti-β-actin monoclonal antibody (Wako Pure Chemical Co., Osaka, Japan) diluted in Can Get Signal solution 1 (Toyobo, Osaka, Japan), after which the membranes were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA).