Figure 1: Structure of the COX2 target protein.

COX2 Target Introduction

Protein Function

• COX2 is a key enzyme in the process of prostaglandin biosynthesis, and it acts as both a dioxygenase and a peroxidase, mediating the formation of prostaglandins from arachidonic acid and playing a unique role in the inflammatory response.
• COX2 is inducible and regulated by specific stimuli, such as infection and inflammation, to produce prostaglandins, whereas its isoform COX1 is constitutively expressed and rapidly produces prostaglandins under stress stimuli.
• In endothelial cells, COX2 can convert docosapentaenoic acid (DPA) into resolvins (RvTs), activating the phagocytic function of macrophages during bacterial infection.
• COX2 is the target of nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin and ibuprofen.
• Inhibiting COX2 can quickly reduce inflammation, pain, and fever reactions. Long-term use of these drugs can reduce fatal thrombotic events, as well as the development of colon cancer and Alzheimer's disease.

Protein Characteristics

• COX2 may require stimulation to be detected in specific cell lines and tissues. We recommend using a positive control as a reference when performing detection.

Protein Expression

• It can be upregulated in response to IL1β or LPS.
• The expression of COX2 varies in different tissues and may need to be optimized based on the tissue cells used in the experiment.

Protein Localization

• COX2 is localized to the nuclear membrane, mitochondrial membrane, and endoplasmic reticulum.

Figure 2: COX2 ICC experimental result image, Anti-COX2/Cyclooxygenase 2 antibody [EPR12012] (ab179800). Green: COX2, Red: Tubulin.

Isoforms & Post-translational modifications

• Human (P35354): 69 kDa (predicted)
• Mouse (Q05769): 69 kDa (predicted)
• Rat (P35355): 69 kDa (predicted)
• Presence of disulfide bonds and N-glycosylation modifications.
• iNOS can mediate S-nitrosylation of COX2 cysteine residues, in addition to Cys-561, S-nitrosylation can also occur on different cysteine residues.
• SPHK1 mediates acetylation of COX2 Ser-565, during neuroinflammation, this acetylation promotes the secretion of specialized pro-resolving mediators (SPM) from neurons, leading to an increase in phagocytic microglial cells.

WB experiment tips

Precautions

• Due to the glycosylation modification of COX2, the band size in WB detection may be different from the predicted value.
  Some cells may need stimulation, such as LPS treatment of Raw 264.7 cells, to detect COX2.

Positive control

• A549, HeLa cell lysate.

Negative control

• Human PTGS2 (COX2 / Cyclooxygenase 2) knockout HeLa cell line (ab255420) / cell lysate (ab263795).

Example of results

Figure 3: WB-Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (ab179800).

Lane 1: A549 cell lysate.
Lane 2: U-87MG cell lysate.
Lane 3: Wild-type HeLa cell lysate.
Lane 4: COX2 knockout HeLa cell lysate.

Result description: COX2 (green), GAPDH (red).
Predicted band size: 69 kDa.

Figure 4: WB-Anti-COX2 / Cyclooxygenase 2 antibody [EP1978Y] (ab62331)

Lane 1: Untreated Raw 264.7 lysate.
Lane 2: LPS-treated Raw 264.7 lysate.

Predicted band size: 69 kDa
Detected band size: 72 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Transfer membrane:

1. We recommend using Ponceau S staining after transfer to check for successful transfer.
2. We recommend not cutting the membrane and keeping the whole membrane or at least a portion of 50-100 kDa for antibody incubation.

Antibody incubation:

1. Please choose the optimal antibody working concentration according to the product manual.

References

1. Sangwon F Kim, Daniel A Huri, Solomon H Snyder. Inducible nitric oxide synthase binds, S-nitrosylates, and activates cyclooxygenase-2. Science. 2005 Dec 23;310(5756):1966-70. doi: 10.1126/science.1119407.
2. Cornelia M Ulrich, Jeannette Bigler, John D Potter. Non-steroidal anti-inflammatory drugs for cancer prevention: promise, perils and pharmacogenetics. Nat Rev Cancer. 2006 Feb;6(2):130-40. doi: 10.1038/nrc1801.
3. Nasser Hashemi Goradel, Masoud Najafi, Eniseh Salehi, Bagher Farhood, Keywan Mortezaee. Cyclooxygenase-2 in cancer: A review. J Cell Physiol. 2019 May;234(5):5683-5699. doi: 10.1002/jcp.27411. Epub 2018 Oct 20.
4. Jan Korbecki, Rafał Bobiński, Mieczysław Dutka. Self-regulation of the inflammatory response by peroxisome proliferator-activated receptors. Inflamm Res. 2019 Jun;68(6):443-458. doi: 10.1007/s00011-019-01231-1.
5. Michael J Lucido, Benjamin J Orlando, Alex J Vecchio, Michael G Malkowski. Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry. Biochemistry. 2016 Mar 1;55(8):1226-38. doi: 10.1021/acs.biochem.5b01378. Epub 2016 Feb 19.