Phosphorylated iκbα

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).

Denatured protein samples of liver tissue or cell lysates were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred onto 0.2 μm PVDF membranes (Merck KGaA, Darmstadt, Germany) and blocked with 5% skim milk for at least 1 h at room temperature. Next, the membranes were incubated overnight at 4°C with the first antibody as indicated: 1:1000 CCN1, 1:1000 NF-κB p65, 1:1000 phosphorylated NF-κB p65, 1:1000, Akt, 1:1000 phosphorylated Akt or 1:1000 GAPDH (Cell Signaling Technology, Danvers, MA, USA), 1:500 IKKα/β, 1:500 phosphorylated IKKα/β, 1:500 IκBα, 1:500 phosphorylated IκBα (Beyotime Biotechnology, Shanghai, China). After washing with PBS-Tween (PBST), the membranes were incubated with HRP-conjugated goat anti-rabbit IgG at room temperature for 1h followed by washing with PBST. Immunoblot images were quantified and analyzed using Image Lab software (Bio-Rad Laboratories, CA, USA).