P ikkα

Plumbagin was purchased from Sigma–Aldrich (St. Louis, MO, USA). Primary antibodies against MMP-2, MMP-9, cleaved caspase-3, Bcl-2, Bax, β-actin, Bad, Bcl-xL were procured from Abcam, USA. TNF-α, NF-κBp65, IκBα, p-IκBα, p-IKKα, IKKα p-IKKβ, IKKβ, and horseradish peroxidase-labeled IgG secondary antibodies were procured from Cell Signaling Technology Inc. (Danvers, MA, USA). ELISA kits for determination of cytokines -TNF-α, IL-1β and IL-6 were obtained from Biolegend (San Diego, CA, USA). Cell lysis buffer for Western blotting analysis was purchased from Beyoime Institute of Biotechnology (Beijing, China). All the other chemicals used for analysis were from Sigma–Aldrich, if otherwise are specified.

Liver homogenates containing 30 μg of whole cell lysate protein or 10 μg of nuclear protein were separated by 10 or 12% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, the blot was probed with primary antibodies for p50, p65, IκBα, IKKα, IKKβ, PCNA and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and cleaved caspase-3, Bcl-2, Bax, p-IKKα and p-IKKβ (Cell Signaling Technology, Beverly, MA, USA). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology) was used as a secondary antibody.

Tissues and cells were homogenized in 10% (wt/vol) hypotonic buffer (25 mM Tris-HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid, 5 μg/mL leupeptin, 1 mM Pefabloc SC, 50 μg/mL aprotinin, 5 μg/mL soybean trypsin inhibitor, 4 mM benzamidine) to yield a homogenate. Then, the final supernatants were obtained by centrifugation at 12,000 rpm for 20 min. Protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin as a standard. Then, the same amount of total protein was subjected to 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting using the following antibodies (1:1,000): rabbit anti-TLR4, p-IkBα, p-IKKα, p-NF-κB, NF-κB, SOD1/2, XO, Nrf2, Keap1, HO-1, GAPDH (Cell Signaling Technology, Inc., Massachusetts, MA, USA), and NQO1 (Abcam). Western blot bands were observed using GE Healthcare ECL Western Blotting Analysis System and exposed to X-ray film of Kodak. Every protein expression level would be defined as the gray value (Version 1.4.2b, Mac OS X, ImageJ; National Institutes of Health, Bethesda, MD, USA) and standardized to housekeeping genes (GAPDH) and expressed as a fold of control.