Nf κb p65

MIN6 cells were seeded in six‐well plates and treated with the combination of IFN‐γ (100 ng/ml) and LIGHT (5 μg/ml) for 0, 0.5, 1, 12, 24 h or for the indicated times. In some experiments, cells were pre‐incubated with or without the NF‐κB inhibitor PDTC for 1 h and then treated with or without IFN‐γ (100 ng/ml) plus LIGHT (5 μg/ml) for 12 h. Antibodies against cytochrome c (BD Pharmingen, Franklin Lakes, NJ, USA), COX4 (BD Pharmingen), NF‐κB p65 (Beyotime Institute of Biotechnology), Bcl‐2 (Cell Signalling, Danvers, MA, USA), Bcl‐xL (Beyotime Institute of Biotechnology, Beijing, China), Bax (Beyotime Institute of Biotechnology), Bak (Beyotime Institute of Biotechnology), caspase‐9 (Beyotime Institute of Biotechnology), PARP (Beyotime Institute of Biotechnology), caspase‐3 (Cell Signalling, Danvers, MA, USA) and cleaved caspase‐3 (Cell Signalling, Danvers) were used to analyse the expression of proteins by Western blot as previously described 5.

Brevilin A (BVA) was provided by PUSH Bio-Technology (Chengdu, China, Figure 1A, the purity ≥98.0%). LPS (E. coli 0111:B4) was purchased from Sigma (Shanghai, China). Murine IFNγ and TNFα were obtained from SinoBiological (Beijing, China). Curcumin, BAY 11-7082, MG132, and LY294002 were purchased from Beyotime (Shanghai, China). PD98059, SB203580, and SP600125 were provided by Selleck (Shanghai, China). Primary antibodies against inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), poly [ADP-ribose] polymerase 1 (PARP1), GAPDH, and Tubulin were obtained from ProteinTech (Wuhan, China). The antibodies against IKKα/β, phosphorylated-IKKα/β, IκBα, phosphorylated-IκBα, NF-κB p65, and Flag were purchased from Beyotime (Shanghai, China). Extracellular regulated protein kinases (ERK), phosphorylated-ERK, c-Jun N-terminal kinases (JNK), phosphorylated-JNK, p38, phosphorylated-p38, RAC-alpha serine/threonine-protein kinases (Akt), and phosphorylated-Akt were provided by Signalway Antibody (Baltimore, United States).

The spleen tissues were ground, and the supernatant was collected by centrifugation. The protein concentration was determined using a BCA assay kit (Solarbio, Beijing, China). The protein (30 μg) was then separated using a 12% SDS-PAGE gel, and a western blot was performed as described in our previous studies (20 (link), 24 (link)). The primary antibodies used were as follows: GAPDH (AF1186, 1:1000), TLR 4 (AF8187, 1:1000), NF-κB p65 (AF0639, 1:1000), IKKα (AF0198, 1:1000), IKKβ (AF7200, 1:1000), IκBα (AF5204, 1:1000), and p-IκB (AF1870 1:1000) were purchased from Beyotime. JNK (#9252, 1:1000), p-JNK (#4668, 1:1000), ERK (#4695, 1:1000), p-ERK (#4370, 1:1000), P38 (#8690, 1:1000) and p-P38 (#4511, 1:1000) were purchased from Cell Signaling Technology.