Tumor necrosis factor (tnf)

Equal amounts (30 μg) of protein extracts from brain of the animals (n = 6, for each group) were loaded and separated by SDS-PAGE using 7% or 12% acrylamide gradients. The membranes were incubated with monoclonal antibodies against NAPH oxidase (NOX)-I (1:1500, Sigma), NOX-II (1:500, Sigma), IL-1β (1:1000, proteintech), r-H2A (1:1000 Abcam), MCP-1 (1:1000, Millipore), TNF-⍺ (1: 1000, Cell Signaling) were used. Signals were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse, goat anti-rat or goat anti-rabbit IgG. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution overnight, followed by a washing procedure carried out three times within 15 minutes. The nitrocellulose membranes were then incubated with the second antibody solution for one hour at room temperature. The washing procedure was repeated three times within 15 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences), which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP).

For the CYPJ and TAB2 colocalization analysis, plasmids encoding GFP-CYPJ and Flag-TAB2 were co-transfected into HeLa cells for 24 h. Mouse anti-Flag antibody was used as a primary antibody and Alexa Fluor 594-conjugated anti-mouse IgG (Zhongshan Golden Bridge Biotechnology) as a secondary antibody. The nucleus was stained with DAPI (KeyGEN Biotechnology). For the P65 nuclear localization analysis, HeLa cells were transfected with GFP-CYPJ for 24 h and then stimulated with TNF (15 ng ml⁻¹, Cell Signaling Technology) for 15 min or unstimulated as control. The nucleus was stained with DAPI (KeyGEN Biotechnology). The cells were then washed three times with PBS, fixed in 4% paraformaldehyde for 20–30 min at room temperature and permeabilized with 0.2% Triton X-100 for 5 min. After blocking in 5% BSA for 30 min, the cells were incubated for 2 h with an anti-p65 monoclonal antibody at 4 °C. After washing with PBS for three times, the cells were incubated for 45 min with Alexa Flour 594-conjugated anti-mouse IgG antibody. The final result was observed by using laser confocal fluorescence microscopy (ZEISS LSM880, Germany).

Recombinant human proteins and inhibitors were purchased as follows: TNF and EGF from Cell Signaling Technology (Beverly, MA, USA), TGFα from Raybiotech Inc. (Norcross, GA, USA), and MK2206 (Akt inhibitor) and PD98059 (Erk inhibitor) from Cayman Chemical Company (Ann Arbor, MI, USA). Antibodies were purchased as follows: NF-κB family members, IκB, EGFR, Erk1/2, Akt, and their phosphorylated forms such as pIκB (Ser32/36), pEGFR (Tyr1173), pErk1/2 (Thr202/Tyr204) and pAkt (Ser473) from Cell Signaling Technology (Beverly, MA, USA); and ErbB isoforms, p65, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A customized PCR array for chemokines, SYBR^® Green Master Mix and RNeasy Mini Kit came from SABiosciences in Qiagen (Frederick, MD, USA). TGFα ELISA kits were from Raybiotech Inc. (Norcross, GA, USA). Chemiluminescent detection kits were from GE Healthcare (Piscataway, NJ, USA). Antisense and sense oligonucleotides were obtained from Eurofins MWG Operon (Huntsville, AL, USA). Lipofectamine 2000 and all liquid culture media were acquired from Invitrogen (Grand Island, NY, USA). The siRNAs for control, Akt and Erk were purchased from Cell Signaling Technology (Beverly, MA, USA). The Luciferase Reporter Assay System was obtained from Promega (Madison, WI, USA).

We carried out the western blot analysis based on the standard procedure in previous reports [18 (link)]. Antibodies for SOD1 (1 : 1000, catalog no.2770), TNF (1: 1000, catalog no.3707), and GAPDH (1: 1000, catalog no.8884) were purchased from Cell Signaling Technology. GAPDH was used as an endogenous control.