Nf κb

Frozen brain cortical tissues were homogenized in ice-cold lysis buffer [31 (link)], and clarified lysates were processed for SDS-polyacrylamide gel electrophoresis under reducing conditions with proteins transferred to nitrocellulose membranes. The membranes were routinely stained with the Ponceau S dye (Sigma-Aldrich, St-Louis, MO, USA) for the rapid and reversible detection of protein bands. Protein expression was detected using specific primary antibodies. Antibodies raised against Bcl2, cleaved caspase 3, FOXO3a, pVHL, acetyl-α-tubulin and phospho-p65Rel (Ser 536) were purchased from Cell Signaling Technology. (Danvers, MA, USA); NF-κB from Epitomics (Burlingame, CA, USA); eNOS from BD Biosciences (Franklin Lakes, NJ, USA); VEGF, ALDH2, SIRT2, β-actin, GAPDH and α-tubulin from Abcam; and HIF-1α from Cayman Chemicals (Ann Arbor, MI, USA). All antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and visualized by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA). Quantitation of the protein bands was performed by volume densitometry using ImageJ software.

The protein was separated with 10% SDS-PAGE and transferred to a membrane. Western blot analysis was performed as described previously, using COMT, GRP75, GRP78, CAHIII, Nrf2, PPAR, CYPOR, Cystathionine gamma-lyase, ubiquitin (Santa Cruz), Catalase, c-Myc, NF-κB, HIF-1, HO-1 (Epitomics), GSTP (Bethyl Lab), Endoplasmin, p53, AKT (Cell Signal), NQO1 (Proteintech), α-SMA, β-actin and anti-DNP (Sigma) overnight at 4°C., followed by incubated with HRP-labeled secondary antibody. Enhanced chemiluminescence was used for signal detection. Expression of β-actin was used to control for equal gel loading [54 (link)].

DHM (purity ≥99.0%) and TAA (purity ≥99.0%) were purchased from Sigma. Hematoxylin-eosin (H and E), TUNEL and Masson stain kits, and commercial assay kits for ALT and AST, SOD, GSH, MDA were obtained from the Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The immunofluorescent staining kits and the enhanced chemiluminescence (ECL) kit were purchased from Beyotime science and technology Co., Ltd. (Beijing, China). The antibody of rabbit monoclonal α-SMA, TGF-β1, CYP2E1, Cleaved Caspase-3, Caspase3, Cleaved Caspase-9, Caspase-9, IκB kinase α (IKK-α), IκB kinase β (IKK-β), p-IKKα, p-IKKβ, inhibitor of IκBα (IκBα), p-IκBα, NF-κB, p-NF-κB, PI3K, p-PI3K, Akt, p-Akt, B-associated X (Bax), Bcl-2, β-actin and secondary antibodies for western blot were all obtained from Abcam (Cambridge, United Kingdom).