Rabbit polyclonal anti cox2 antibody

Curcumin was purchased from Cayman Chemical (Ann Arbor, MI, USA). Phosphate‐buffered saline (PBS, pH 7.4), paraformaldehyde (PFA), Triton X‐100 and BCA assay kits were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Thioflavin‐S was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Cell lysis buffer and phenylmethylsulfonyl fluoride (PMSF) were purchased from Beyotime Biotechnology (Shanghai, China). Primary antibodies used for immunofluorescence staining and Western blot, including goat polyclonal anti‐GFAP antibody, rabbit polyclonal anti‐COX2 antibody, rabbit monoclonal anti‐HMGB1 antibody, rabbit polyclonal anti‐RAGE antibody, mouse monoclonal anti‐TLR4 antibody, rabbit monoclonal anti‐NF‐κB p65 antibody and mouse polyclonal anti‐β‐actin antibody were purchased from Abcam (Cambridge, UK), Cell Signaling Technology (CST, Danvers, MA, USA) or Bioworld (St. Louis Park, USA). Secondary antibodies, including DyLight 488 AffiniPure Donkey Anti‐Goat IgG, HRP‐labelled rabbit anti‐goat, HRP‐labelled goat anti‐rabbit and HRP‐labelled goat anti‐mouse, were obtained from EarthOx (Millipore, Burlington, MA, USA) or Fdbio Science (Hangzhou, China).

For analysis of COX-1 and COX-2 expression, cells and tissues were lysed with Tris·HCl buffer [50 mM, pH 7.4, NaCl (300 mM), EDTA (2 mM)], NP-40 (1%), PMSF (1 mM) and inhibitor cocktail (Roche). The protein samples were boiled for 5 min in reducing Laemmli buffer supplemented with 5% 2-mercaptoethanol. Equal amounts of protein were subjected to electrophoresis (4-20% Tris·HCl gel, BioRad) and blotted onto PVDF membrane (BioRad). Immunostaining of COX-1 was performed with goat polyclonal anti-COX-1 antibody and immunostaining of COX-2 with rabbit polyclonal anti-COX-2 antibody (both 1:1000, abcam, Cambridge, UK). The secondary anti-goat and anti-rabbit horseradish peroxidase coupled antibodies (both 1:2000, Cell Signaling, Cambridge, UK) were visualized with enhanced chemoluminescence (ECL+, GE Healthcare, UK) as recommended by the supplier. Equal protein loading was controlled using GAPDH specific antibody (1:1000, Cell Signaling) and secondary anti-rabbit antibody linked to horseradish peroxidase.

Immunofluorescence staining assay was applied to observe the expression and location of COX-2 in HBMSCs cultured with different media. Briefly, after HBMSCs were incubated with or without CS ionic products in confocal dishes for 24 h, the cells were washed two times with DPBS and fixed by 4% paraformaldehyde for 15 min. Then, the cells were permeabilized with 20% Triton for 5 min at room temperature and blocked with DPBS containing 1% BSA (Sigma) for 1 h at 37°C, followed by being incubated with primary antibody solution containing rabbit polyclonal anti-COX2 antibody (1:100, abcam, USA) for 2 h at 37°C and overnight at 4°C. Then, the cells were washed two times with DPBS and incubated with secondary antibody solution containing Alexa 488 goat anti-rabbit IgG antibody (1:1000, abcam) for 1 h at 37°C. Cell nuclei were revealed with 5 μg/ml 4,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Finally, the cells were observed with a laser confocal microscope (Leica SP5, Germany), and images were taken with a CCD camera (Leica DFC 420C). Three images were taken per sample, and the mean fluorescence intensity of each image was measured with ImageJ software.

The siRNA oligonucleotides were purchased from either Qiagen (Valencia, CA) or Dharmacon (Boulder, CO). Histidine-lysine co-polymer (HKP) was provided by Biopolymer lab at the University of Maryland or purchased from Ambio Pharma Inc. (Shanghai, China). Primers PCR were purchased from Qiagen (Germantown, MD) or Genepharm (Suzhou, China). Rabbit polyclonal anti-alpha smooth muscle Actin (α-SMA) antibody, Rabbit polyclonal anti-COX2 antibody, Rabbit monoclonal anti-TGF-β1, Rabbit Monoclonal anti-human MHC class 1 antibody, Rabbit polyclonal antibody to CD31, and Rabbit polyclonal antibody to VEGF were purchased from Abcam Corporation (Iowa, USA). Rabbit monoclonal anti-SMAD2/3, anti-p-SMAD2/3 and anti PAI-1 antibody were purchased from Cell Signaling Technology (Danvers, MA), The Bio-Rad iScript reverse transcription kit and IQ Sybr Green Supermix reagents (Hercules, CA) were used for qRT-PCR performed with Bio-Rad MyiQ Thermal Cycler. COX2 inhibitor Celecoxib (Catalog No.S1261) and TGFβ receptor I (TBR1) antagonist Galunisertib (LY2157299) (Catalog No.S2230) were purchased from Selleck Chemicals (Shanghai, China).