In-cell Westerns used to Demonstrate Potent Anti-inflammatory Activity of Mosquito Saliva Protein

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Both the Azure 300 and Sapphire Biomolecular Imager, along with fluorescent blocking buffer and secondary antibodies from Azure Biosystems, were used in a recent publication revealing anti-inflammatory activity of a mosquito saliva protein. Shrivastava et al from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (NIH) in Rockville, MD, employed both instruments in their study demonstrating how protein Alboserpin inhibits pro-inflammatory activities of the coagulation cascade protein factor Xa (FXa).

Since the release of this publication, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins.

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By traditional and in-cell Western blots, Alboserpin was shown to block the FXa-induced increase in phosphorylated ERK1/2, indicating it blocks FXa-induced ERK1/2 signaling. The chemiluminescent blots were imaged using the Azure 300, while the in-cell Western blots (Figure 1D) were imaged on the Sapphire with fluorescent blocking buffer and fluorescent secondary antibodies.

Figure 1D from Shrivastava et al (2022), Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling in vitro and in vivo
Figure 1D from Shrivastava et al (2022), Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling in vitro and in vivo. Licensed under CC BY 4.0. In-Cell Western blots conducted using Azure fluorescent secondary antibodies and imaged on the Azure Sapphire Biomolecular Imager demonstrate a decrease in Fxa-induced phosphorylation of EKR 1/2 in the presence of Alboserpin.

These results from the NIH provide additional insight into the mechanisms mosquito vectors employ to avoid host responses during blood feeding. The authors indicate further research is needed to examine the role Alboserpin might play in pathogen transmission.

The new Sapphire FL is the ultimate biomolecular imager for flexibility. With customizable and user-changeable laser and filter modules, the Sapphire FL easily adapts to a lab’s changing needs and advancing research.

Alboserpin and its role on FXa

FXa is known to trigger inflammation by signaling through protease-activated receptors (PARs). In their recently published work, Shrivastava et al demonstrate that Alboserpin has anti-inflammatory activity, in vitro and in vivo, due to its inhibition of FXa activities. Across multiple experiments, Alboserpin was shown to prevent numerous FXa-induced processes including FXa-induced release of proinflammatory cytokines, increased expression of multiple PARs and other proteins (specifically PAR-1, PAR-2, PAR-3, NF-kB, ICAM, and VCAM-1), induced activation of NF-kB, and cleavage of PAR-2.

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What are Aedes mosquitos? Why is their saliva important?

Aedes mosquitos are an important vector for arthropod-borne (arbo) viruses such as Zika, dengue, and chikungunya viruses. Arbo viruses are responsible for substantial disease burden, morbidity, and mortality among human populations world-wide. These viruses can be introduced into a host when the mosquito pierces the skin to draw blood and releases saliva at the site of the bite.

The mosquito saliva contains proteins that prevent hemostasis (a process that involves reduced blood flow), coagulation, and clot formation to prevent blood loss at the site of an injury. The Aedes albopictus mosquito has an anticoagulant called Alboserpin in its saliva. Alboserpin is a serine protease inhibitor known to demonstrate competitive, reversible, and high-affinity binding to FXa, an important component of the coagulation cascade.

More about in-cell Westerns: History behind In-cell Westerns

In addition to chemiluminescent and multicolor fluorescent imaging of blots and multi-well plates, the Sapphire Biomolecular Imager can carry out densitometry, white light, phosphor, and near infrared imaging of blots, gels, tissues, and more. Learn more about the Sapphire Imager and how Azure can support your research by clicking here.

Shop Reagents used by NIH in this study
  1. Shrivastava G, Valenzuela-Leon PC, Chagas AC, et al. Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling in vitro and in vivo. ImmunoHorizons. 20022;6(6):373-383.

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