Cox 2 antibody

All reagents were high grade and were purchased from Sigma with the following exceptions. RPMI, DMEM, Calcein and other culture reagents were purchased from Invitrogen Inc (Grand Island, NY, USA) and the UCSF cell culture facility (UCSF, San Francisco, CA). Fetal bovine Serum Defined (FBS) was purchased from Hyclone Laboratories (Logan, UT, USA). Selective HSP90 inhibitors: Geldanamycin, 17-AAG, and radicicol, and BIIB021 were purchased from Calbiochem (San Diego, CA). Lipopolysaccharide (LPS, Escherichia coli, O26:B6) was purchased from Sigma (St Louis, MO). Drugs were dissolved in DMSO or ethanol and stored at −20°C and either used (final concentration of vehicle 0.1% (v/v or dried down and resuspended in PBS/0.1% bovine serum albumin (BSA). HSP antibodies were purchased from Assay design (Enzo Life sciences), for MAP stress activated kinase anti–phospho-JNK/SAPK mAb (#4668) were from Cell Signaling Technology (Danvers, MA); anti–NF-kBp65 (# SC-8008), anti–IkBα (# SC-1643) and respective horseradish peroxidase–coupled secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA) and antibodies against mouse HSP70i (#SPA-810) from Enzo Life sciences, iNOS (# 61043), was from BD Biosciences (BD Biosciences, Lexington, KY); COX-2 antibody is from Cayman Chemical Company (Ann Arbor, MI (# 160106).

Western blot was used to measure the protein expression of COX-2 using a standard method prescribed previously (27 (link)). In short, the gastric fundus and antrum were homogenized on ice in lysis buffer supplemented with 1/100 proteinase inhibitor cocktail (Sigma Aldrich, St Louis, MO). The lysis buffer was made with following ingredients: 20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na₃VO4, 1 ug/mL leupeptin. Equal amount of total proteins were separated on 4%–12% Bis-Tris NuPAGE Gels (Life Technologies, Carlsbad, CA). Then they were transferred to nitrocellulose membranes (BioRad, Hercules, CA) and blocked for 1 hour in blocking buffer (LI-COR Biosciences) at room temperature. The membranes were incubated with COX-2 antibody (1/500 dilution, Cayman Chemical, Ann Arbor, MI) overnight at 4°C. Then Alexa Fluor 680 goat anti-rabbit IgG (1/5000 dilution, Invitrogen) was applied for 1 hour at room temperature. The analysis was finalized by Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

Anti-β-amyloid precursor protein (AβPP) and anti-occludin antibodies were purchased from Zymed Laboratories (San Francisco, CA). Anti-rabbit (goat), anti-goat (bovine), anti-rat (goat), and anti-mouse (bovine) horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). CD36 and actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Elite Vectastain ABC reagents, Vector VIP, biotinylated anti-rabbit, anti-mouse, and anti-rat antibodies were purchased from Vector Laboratories Inc. (Burlingame, CA, USA). Anti-human and anti-mouse CD68 antibodies were purchased from AbD Serotec (Oxford, UK). BACE antibody was purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). COX-2 antibody was purchased from Cayman Chemical (Ann Arbor, MI, USA). Aβ clone 4G8 was purchased from Covance (Emeryville, CA, USA). Anti-amyloid beta precursor protein antibody (Y188) was purchased from Abcam (Cambridge, MA, USA). The PHF-1 anti-phosphoSer396/404 mouse monoclonal antibody was a generous gift from Dr. Peter Davies, Albert Einstein College of Medicine.