Cyclooxygenase 2 cox2

RIPA lysis buffer with 1 mM protein phosphatase inhibitor was added into rCHs for protein extraction. The lysate was collected and centrifuged for 10 min at 12,000 r/min at 4°C. A BCA protein assay kit (Thermo Scientific) was used to determine the concentration of protein. The proteins were separated by 10% SDS-polyacrylamide gels, and then transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The block solution was the TBS (Tris-HCL Buffer Solution) containing bovine serum albumin (0.5%) (BSA, Sigma-Aldrich, Darmstadt, Germany) and Tween-20 (0.1%) (Bio Froxx, Guangzhou, China). The membranes were blocked with block solution for 1 h at room temperature, incubated with antibodies against matrix metalloproteinase 13 (MMP13, 1:1000, GTX100665, GeneTex, CA, USA), CollagenII (1:1000, ab34712, Abcam, Cambridge, UK), cyclooxygenase-2 (COX2, 1:1000, #12282, Cell Signaling Technology, Danvers, USA), TLR2 (1:1000, #29071, SAB, Nanjing, China), p-NF-κB (1:1000, #3033T, Cell Signaling Technology, Danvers, USA), NF-κB (1:1000, #8242T, Cell Signaling Technology, Danvers, USA) at 4°C overnight. Next, the TBST was used to wash membranes and the membranes incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. The ChemiDocXRS + Imaging System (Tanon, Shanghai, China) was used to detect the signals. The protein bands were quantitatively analyzed by ImageJ.

PACA was synthesized and characterized by NMR and MS as previously described [22 (link), 23 (link)]. Cell culture medium, fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against iNOS, cyclooxygenase-2 (COX-2) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and PPAR-γ was purchased from Cell Signaling Technology (Boston, MA, USA). The antibodies against Arg1, ED2 and CD163 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against ED1 and CD68 were purchased from Abcam (Cambridge, Massachusetts, UK). Anti-mouse CD80 FITC was obtained from BD Biosciences (San Diego, CA, USA). Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody were purchased from Life Technologies (Carlsbad, CA, USA). Anti-rabbit HRP-conjugated IgG secondary antibody and other chemicals including GW9662, a specific PPAR-γ inhibitor, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

ME was purchased from the National Institute for Food and Drug Control (Beijing, China; purity, ≥99.5%). Dulbecco's minimum essential medium (DMEM), penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). 4-[3-(4-Iodophenyl)-2-4(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) was obtained from Dojindo (Kumamoto, Japan). Specific antibodies against phospho-Lyn, Lyn, phospho-Syk, Syk, phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK, JNK, cytosolic phospholipase A₂ (cPLA₂), phospho-cPLA₂, cyclooxygenase-2 (COX-2), and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Specific antibodies against phospho-5-lipoxygenase (5-LO) and 5-LO, and enzyme immunoassay (EIA) kits for prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂), leukotriene B₄ (LTB₄), and leukotriene C₄ (LTC₄) were purchased from Cayman Chemical (Ann Arbor, MI, USA). The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-4 were obtained from Bangyi Technologies Inc. (Shanghai, China). Dinitrophenyl- (DNP-) IgE was obtained from Sigma-Aldrich (St Louis, MO, USA), and DNP-bovine serum albumin (BSA) was obtained from Biosearch Technologies Inc. (Novato, CA, USA). All other chemicals were of analytical grade and were purchased from Sigma-Aldrich.

Immunohistochemical staining was performed according to a previously described method (Gu et al. 2015 (link)). The sections were reacted with specific primary antibodies for Aβ (1:300; Abcam, Inc., Cambridge, MA, USA), glial fibrillary acidic protein (GFAP) (1:300; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), ionize calcium-binding adapter molecule 1 (IBA-1) (1:300; Abcam, Inc., Cambridge, MA, USA), inducible nitric oxide synthase (iNOS) (1:300; Novus Biologicals, Inc., Littleton) and cyclooxygenase-2 (COX-2) (1:300; Cell Signaling Technology, Inc., Beverly, MA, USA). Tissues were then incubated with the corresponding conjugated secondary antibodies: goat anti-rabbit, goat anti-mouse, or donkey anti-goat IgG-horseradish peroxidase (HRP) (1:5000; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA). The sections were evaluated under a light microscope (Microscope Axio Imager.A2, Carl Zeiss, Oberkochen, Germany) (×50 or ×200 or ×400).