Cox 2

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Germany

COX-2 is a laboratory assay used to measure the activity of the cyclooxygenase-2 enzyme. Cyclooxygenase-2 is an inducible enzyme that is responsible for the production of prostaglandins in response to various stimuli, such as inflammation and tissue damage. The COX-2 assay provides a quantitative assessment of the enzyme's activity, which is useful for researchers studying inflammatory processes and the development of anti-inflammatory drugs.

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Lab products found in correlation

195 protocols using cox 2

Western Blot and qRT-PCR Analysis of Tumor Proteins

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Proteins within tumour tissues and HUVEC cells were extracted using a Boster Kit (Bosterbio, CA, USA) according to the manufacturer’s instructions. Western blotting was performed as previously described [20 (link)]. The antibody, which was raised against the Cyp2c44’s IGRHQPPSMKDKMKC peptide (GenScript), was generated according to previous studies [13 (link), 16 (link)]. Other antibodies used in this study are as follows: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bosterbio), β-actin (Bosterbio), Cyp2c9 (Abcam, Cambridge, UK), COX1 (Santa Cruz, CA, USA), COX2 (Santa Cruz), Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) (Santa Cruz), ERK1/2 (Santa Cruz), P-AKT (Abcam), AKT (Abcam), and CD31 (Abcam).
For qRT-PCR, RNA from tumours was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and reverse-transcribed using the M-MLV First-Strand cDNA Synthesis Kit (Invitrogen) [15 (link)]. The mRNA levels of target genes were quantified by qRT-PCR using Power SYBR Green PCR Master Mix (Invitrogen) with the primers listed in Additional file 1: Table S1. GAPDH served as an internal control and the results were analysed using the 2^-ΔΔCt method.

Wu L., Wang W., Dai M., Li H., Chen C, & Wang D. (2019). PPARα ligand, AVE8134, and cyclooxygenase inhibitor therapy synergistically suppress lung cancer growth and metastasis. BMC Cancer, 19, 1166.

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Grape Seed Extract Characterization and Bioactivity

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Grape seed extract (GSE, ORAC value 9000–13,000 μmole Trolox equivalents/g, total phenolic content > 85 % gallic acid equivalents) was a generous gift from San Joaquin Valley Concentrates (Fresno, CA). We had previously characterized the GSE used in this study using UPLC-MS and we detected presence of (+)-catechin and (−)-epicatechin monomers and their oligomers, and their gallate derivatives similar to other published papers [24 (link), 25 (link)]. The GSE used in this study lacks resveratrol (RSV) as described earlier [10 (link)]. BrdU Cell Proliferation Assay Kit was obtained from Cell Signaling Technology (Danvers, MA). Antibodies for PARP and cleaved PARP, p53, pGSK3β, Bax, Bcl-2, β-actin, β-catenin, cyclin D1, c-Myc, COX-2 and topoisomerase-2β (Topo ii b) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cytochrome C was obtained from Cell Signaling Technology (Beverly, MA). All other chemicals including RSV were obtained from Sigma (St. Louis, MO).

Reddivari L., Charepalli V., Radhakrishnan S., Vadde R., Elias R.J., Lambert J.D, & Vanamala J.K. (2016). Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis. BMC Complementary and Alternative Medicine, 16, 278.

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Intestinal Inflammatory Protein Analysis

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Immunoblotting was performed as described previously [16 (link)]. Briefly, dissected intestinal were homogenized and lysed with lysis buffer. Protein extract was separated on SDS-PAGE and transferred to polyvinylidenedifluoride (PVDF) membrane and probed with the following antibodies: IL-6, IL-1β, TNF-α, PGE2, COX-2, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA). Band intensity was determined by densitometry analysis using the ImageJ analysis system.

Park S.A., Lee G.H., Hoang T.H., Lee H.Y., Kang I.Y., Chung M.J., Jin J.S, & Chae H.J. (2021). Heat-inactivated Lactobacillus plantarum nF1 promotes intestinal health in Loperamide-induced constipation rats. PLoS ONE, 16(4), e0250354.

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Western Blot Analysis of Cellular Proteins

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Cell protein was isolated by using the cell lysis buffer (Beyotime Institute of Bio-technology, China). Western blot analysis was performed as previously reported[22 (link)]. The primary antibodies for Cox-2, Bcl-2, Bax, GAPDH, cyclin D1, cyclin E, and Wee1 were obtained from Santa Cruz Biotechnology (United States). Quantification of optical density was evaluated using Uvitec Alliance software (Eppendorf, Germany) (n = 3 independent experiments).

Guo S.S., Wang Y, & Fan Q.X. (2019). Raddeanin A promotes apoptosis and ameliorates 5-fluorouracil resistance in cholangiocarcinoma cells. World Journal of Gastroenterology, 25(26), 3380-3391.

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Pathway Regulation in BEAS-2B Cells Exposed to B[a]P

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When BEAS-2B cells and its stable transfection cells were cultured in 6-well plates to 50∼60% confluence, the culture medium was replaced with 1% FBS DMEM. After 12 h, the cells were exposed to 8 μM B[a]P for indicated time. Cells were then lysed in 1 × cell lysis buffer plus 1 × phenylmethanesulfonyl fluoride (PMSF). Cellular lysates were centrifuged at 12,000 rpm for 15 min at 4°C, and then separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were blotted onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad, USA) that was probed with each of the following antibodies against β-Actin (Beyotime Institute of Biotechnology, China), SIRT1, β-catenin, COX-2 (Santa Cruz, USA). The protein bands specifically bound by the primary antibodies were detected using horse reddish peroxidase-linked secondary antibody and ECF (Beyotime Institute of Biotechnology, China).

Lu J., Zhang M., Huang Z., Sun S., Zhang Y., Zhang L., Peng L., Ma A., Ji P., Dai J., Cui T., Liu H, & Gao J. (2015). SIRT1 in B[a]P-induced lung tumorigenesis. Oncotarget, 6(29), 27113-27129.

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Immunohistochemical Profiling of Stem Cell Markers

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5 μm fixed sections were incubated with primary antibodies in a hybridization chamber for 1 h at room temperature or overnight at 4°C. The primary antibodies used were DclK1, COX-2, 5-LOX, Ki67, proliferating cell nuclear antigen (PCNA), CD133, CD44, Lgr5, Annexin V and β-catenin procured from Santa Cruz/Abgent/Abcam/Abcam/Cell Signaling. Following primary antibody, sections were incubated for 1 h with anti-mouse/anti-rabbit/anti-goat secondary antibody, then visualized with diaminobenzidine (DAB) and counterstained with H&E for IHC or with DAPI for immunohistofluorescence (IHF). Slides were observed under an Olympus microscope 1X701 and digital computer images were recorded with an Olympus DP70 camera.

Mohammed A., Janakiram N.B., Madka V., Brewer M., Ritchie R.L., Lightfoot S., Kumar G., Sadeghi M., Patlolla J.M., Yamada H.Y., Cruz-Monserrate Z., May R., Houchen C.W., Steele V.E, & Rao C.V. (2015). Targeting pancreatitis blocks tumor-initiating stem cells and pancreatic cancer progression. Oncotarget, 6(17), 15524-15539.

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Western Blot Analysis of Cell Signaling

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Cells were lysed in the M-PER mammalian protein extraction reagent (Thermo, Rockford, IL) containing halt protease inhibitor single-use cocktail (Thermo). The protein concentration was determined by the BCA reagents (Thermo). About 10 μg proteins were denatured and applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis products were transferred to a polyvinyl idenefluoride (PVDF) film and PVDF membranes were then incubated at room temperature with specific primary antibody. After a standard washing, membranes were incubated with horse radish peroxidase (HRP)-labeled secondary antibody. The assay developed using a chemiluminescent substrate. The primary antibodies used in this study included antibodies against β-actin, p-p44/p42 MAPK, p44/p42 MAPK, p-p38 MAPK, p38 MAPK, p-JNK, JNK (Cell Signaling Technology), EV71 VP1 (Abnova) and COX-2 (Santa Cruz Biotechnology). The goat anti-rabbit and anti-mouse HRP-labeled antibodies were obtained from Cell Signaling Technology.

Wang H., Zhang D., Ge M., Li Z., Jiang J, & Li Y. (2015). Formononetin inhibits enterovirus 71 replication by regulating COX- 2/PGE2 expression. Virology Journal, 12, 35.

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Western Blot Analysis of iNOS and COX-2

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Cells were washed in ice-cold PBS, resuspended and lysed by agitation in CelLytic-M Cell Lysis Reagent (Sigma), and centrifuged at 18,000 × g for 10 min at 4°C to yield the resultant supernatant lysate. Western blots were performed as described previously by Poulose et al. (JAFC, 60, 1084–93, 2012), except that 10% polyacrylamide gels were used. Primary antibodies for iNOS (Millipore, Billerica, MA) and COX-2 (Santa Cruz, Dallas, TX) were used at 1:1000 dilutions for incubation overnight at 4°C. Following ECL (enhanced chemiluminescence) development, the optical density of antibody-specific bands was analyzed by the VisionWorks LS image acquisition and analysis software (UVP, Upland, CA).

Rutledge G.A., Fisher D.R., Miller M.G., Kelly M.E., Bielinski D.F, & Shukitt-Hale B. (2019). The effects of blueberry and strawberry serum metabolites on age-related oxidative and inflammatory signaling in vitro. Food & function, 10(12), 7707-7713.

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Western Blot Analysis of COX-2 Expression

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Total protein was extracted from the cultured cells using the PRO‐PREP protein extraction solution (iNtRon Biotechnology), and quantified using DC protein assay kit (Bio‐Rad). After separating 20 μg of protein by SDS‐PAGE, they were transferred on to polyvinylidene difluoride membranes (Millipore). The membranes were then blocked with 5% non‐fat dry milk and incubated with primary antibodies against COX‐2 (1:500; Santa Cruz Biotechnology) and β‐actin (1:1,000, Santa Cruz Biotechnology) at 4°C overnight. Next day, the membranes were incubated with secondary antibody for 1 hr at 20°C and then visualized by chemiluminescence (Advansta, Menlo Park).

Yang S., An J., Park S., Lee J., Chae H., Lee K., Song W, & Youn H. (2021). Enhanced expression of cyclooxygenase‐2 related multi‐drug resistance gene in melanoma and osteosarcoma cell lines by TSG‐6 secreted from canine adipose‐derived mesenchymal stem/stromal cells. Veterinary Medicine and Science, 7(3), 968-978.

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Western Blot Analysis of Signaling Pathways

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Keratinocytes and macrophages were harvested after FxRF and PM or H-PM treatment, and cells were then homogenized with lysis buffer. The supernatants of the lysates were obtained by centrifugation, and protein content was measured using a commercial kit. Lysis buffer containing 30 μg protein was separated on a polyacrylamide gel (12%). The proteins were then transferred onto nitrocellulose membranes. A solution containing 5% nonfat milk was used to block the membranes for 3 h, and the membranes were then incubated with primary antibodies (β-actin, p38 MAPK, P-p38 MAPK, Erk1/2, P-Erk1/2, JNK, P-JNK, iNOS, and COX-2 (Santa Cruz Biotechnology) (4 °C for 8 h), followed by incubation with secondary antibodies (anti-mouse IgG, Santa Cruz Biotechnology) at room temperature for 3 h. A chemiluminescent substrate was employed to develop the blots. The fluorescence images and calculation of band intensity were performed as previously reported [27 ].

Dai Y.L., Jiang Y.F., Lu Y.A., Yu J.B., Kang M.C, & Jeon Y.J. (2021). Fucoxanthin-rich fraction from Sargassum fusiformis alleviates particulate matter-induced inflammation in vitro and in vivo. Toxicology Reports, 8, 349-358.

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