Anti cox 2 antibody

Western blot was performed according to previous studies [24 (link), 32 (link)] to assess inflammation (COX-2) and collagen deposition (α-SMA, MMP-9) to further delineate the mechanism of emodin (SMAD3). Mammalian protein lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract the total tissue protein. The same amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in the diluted primary antibodies overnight and stored at 4°C. The primary antibodies were anti-COX-2 antibody (Santa Cruz, 1 : 200 dilution), anti-SMAD3 antibody (Santa Cruz, 1 : 500 dilution), anti-α-SMA antibody (Santa Cruz, 1 : 200 dilution), and anti-beta-actin (β-actin) antibody (Santa Cruz, 1 : 1000 dilution). The membranes were incubated with the secondary antibody followed by horseradish peroxidase (HRP; Santa Cruz). Then, an enhanced chemiluminescence system (EMD Millipore) was used to detect the bands. The intensity of the bands was calculated using Image-Pro Plus 5.0 software (Media Cybernetics Inc., Rockville, MD, USA).

Briefly, 20 μg of total cell lysates were separated by 12% SDS–PAGE and transferred to a nitrocellulose membrane as described elsewhere (35 (link)). The following primary antibodies were applied: anti-COX-2 antibody (Santa Cruz Biotechnology; sc-1745; 1/100 dilution) and anti-MMP-13 antibody (Abcam; ab39012; 1/6000 dilution) in 3% BSA, overnight at 4°C. Antibody binding signal was detected by chemoluminescence through horseradish peroxidase-linked secondary antibodies. α-Tubulin (Sigma-Aldrich; T5168; 1/5000 dilution) was used for protein loading control. Densitometric measurements were normalized relative to protein presence in IL-1β and TNFα-stimulated chondrocytes using Quantity One software (Bio-Rad) (20 (link)).

Sample cross-sections were deparaffinized and rehydrated with xylene and graded alcohols. After antigen retrieval, the endogenous peroxidase was blocked, and the sections were incubated with primary rabbit anti-TNF-α antibody (1:100 in bovine serum albumin [BSA], ABCAM) or anti-COX-2 antibody (1:200 in BSA, Santa Cruz Biotechnology, USA) followed by incubation with biotinylated goat anti-rabbit antibody (diluted 1:800 in BSA, Santa Cruz Biotechnology, USA). The slides were washed and incubated with the avidin-biotin-horseradish peroxidase conjugate (Strep ABC complex by Vectastain^® ABC Reagent and peroxidase substrate solution), according to the Vectastain protocol (Vector Laboratories, Inc., Burlingame, CA, United States). Immunostaining was visualized with the chromogen 3,3’-diaminobenzidine (DAB). The negative control sections were processed simultaneously. The slides were counterstained with Harry’s hematoxylin, dehydrated in graded alcohol series, cleared in xylene, and coverslipped. TNF-α and COX-2 expressions were blinded scored based on the intensity of the staining, as follows: no staining (0); weak staining (1); moderate staining (2); moderate–intense staining (3); intense staining (4), according to Lima-Júnior and colleagues (13 (link)).

Culture medium DMEM, penicillin, streptomycin, PBS, Trypsin-EDTA and fetal bovine serum (FBS) were purchased from Gibco. MTT, 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), LPS (lipopolysaccharide from E. coli, 0127:B8), Griess reagent, Dexamethasone (Dex) and GW9662 were purchased from Sigma-Aldrich, USA. Anti-Beclin-1, anti-Caspase-1, anti-NF-κB and anti-TNF-α antibodies were purchased from Abcam (MA, USA). Anti-LC-3 antibody was purchased from Cell Signaling while Anti-COX-2 antibody was obtained from Santa Cruz Biotechnology Inc. (Texas, USA). Anti-iNOS antibody was purchased from Thermo Fisher Scientific (MA, USA). Enzyme immunoassay kit for IL-1β, IL-10 and PGE₂ were purchased from R&D system, Inc. (MN, USA) whereas PGD₂ assay kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). AA, LA, ALA and DHA were procured from Nu-Chek-Prep (MN, USA). The 15-LOX metabolites of ALA, 13-(S)-HPOTrE and 13-(S)-HOTrE, were generated (Supplementary Figure S6) essentially employing the methods described earlier for generation of 5-LOX metabolites65 (link)66 (link)67 (link).