Anti tnfr2

Anti-MMP-12, anti-MMP-9, anti-GFAP, anti-MOG, anti-Iba1, anti-TNFα, anti-TNFR1, anti-TNFR2, anti-caspase-3, anti-cytochrome C, anti-AIF, and anti-MBP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-NeuN antibody was obtained from Millipore (Billerica, MA). Anti-glial fibrillary acidic protein (GFAP) was obtained from Dakocytomation (Carpinteria, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Novus Biologicals (Littleton, CO).

Dulbecco’s modified Eagle’s medium (DMEM)/F-12 and fetal bovine serum were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). BioTrace^TM NT membrane was purchased from Pall Life Sciences (Ann Arbor, MI, USA). The enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Perkin Elmer (Waltham, MA, USA). Anti-phospho-EGFR (Tyr¹¹⁷³, Cat#4407), anti-phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸², Cat#9211), anti-phospho-JNK1/2 (Thr¹⁸³/Tyr¹⁸⁵, Cat#9255), anti-phospho-FoxO1 (Ser²⁵⁶, Cat#9461), and anti-phospho-p65 (Ser⁵³⁶, Cat#3033) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH (Cat#MCA-1D4) was purchased from EnCor Biotechnology (Gainesville, FL, USA). AG1478, SP600125, and Tanshinone IIA were purchased from Enzo Life Science (Farmingdale, NY, USA). AS1842856 was obtained from EMD Millipore (Billerica, MA, USA). Helenalin was purchased from Cayman Chemical (Ann Arbor, MI, USA). Recombinant human TNF-α protein was purchased from R&D Systems (Minneapolis, MN, USA). TRIzol reagent, enzymes, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell protein homogenates (25 μg) were separated by electrophoresis in 12% polyacrylamide gels, transferred to nitrocellulose membranes, and blocked overnight with 5% nonfat dry milk. The membranes were washed and incubated in the presence of the following monoclonal antibodies: anti-IL-1R1, anti-IL-1R2, anti-TNFR1, anti-TNFR2, anti-VDR (sc-393998, sc-376247, sc-8436, sc-393614, and sc-13133, respectively; Santa Cruz Biotechnology, CA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, MAB374, Millipore, Milford, MA, USA) overnight at 4°C. Membranes were incubated in the presence of secondary antibody conjugated with horseradish peroxidase (sc-2031, Santa Cruz Biotechnology) for 2 hours at room temperature. The immunoblots were visualized by chemiluminescence using ECL Plus (Amersham Pharmacia, UK).