Mouse anti nfκb p65 polyclonal antibody

MH, lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carbon tetrachloride (CCl₄) was purchased from Shanghai Macklin Chemical Co., Ltd. (Shanghai, China). TNF-α and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were from R&D Systems (Minneapolis, MN, USA). Lipid peroxidation malondialdehyde (MDA) assay kit, total glutathione assay kit, and total superoxide dismutase (SOD) assay kit were from Beyotime Institute Biotechnology (Shanghai, China). TRIzol reagent is from Life Technologies (Grand Island, NY, USA). Mouse anti-NFκB p65 polyclonal antibody and rabbit anti-IκB monoclonal antibody were from Cell Signaling Technology (Boston, MA, USA); rabbit anti-TLR4 monoclonal antibody was from Epitomics, Inc. (Burlingame, CA); rabbit anti-Nrf2 monoclonal antibody, rabbit anti-TREM-1 polyclonal antibody, rat anti-F4/80 monoclonal antibody, rabbit anti-HO-1 polyclonal antibody, rabbit anti-GAPDH monoclonal antibody, and rabbit anti-Lamin B1 monoclonal antibody were from Abcam (Cambridge, MA).

The EBL cells were transfected with siANXA2 or siCtrl and then infected with M. bovis at a MOI of 1000. While the ANXA2-KO cells (KO) and EBL wildtype cells (WT) were also infected with M. bovis at the same condition. Cells were harvested at 0, 4, 8, 12, and 24 hpi and lysed with RIPA lysis buffer containing a proteinase inhibitor cocktail (Thermo Scientific, Rockford, IL), and protein concentrations were determined by the Bradford assay and equalized before loading. Equal amounts of protein samples were separated on 12% SDS-PAGE, transferred to PVDF membranes, and probed with the following primary antibodies: Rabbit anti-ANXA2 polyclonal antibody, Mouse anti-NF-κB p65 polyclonal antibody, rabbit anti-phospho-NF-κB p65 polyclonal antibody, rabbit anti-ERK polyclonal antibody, rabbit anti-phospho-ERK polyclonal antibody, rabbit anti-JNK polyclonal antibody, rabbit anti-phospho-JNK, rabbit anti-p38 polyclonal antibody and rabbit anti-phospho-p38 polyclonal antibody (Cell Signaling Technology, Massachusetts, USA), then followed by washing and incubation with respective HRP-conjugated goat anti-mouse or anti-rabbit IgG (SouthernBiotech). An enhanced chemiluminescence detection system was used to visualize the reactivity, and the band intensity was analyzed using ImageJ software.