For decades researchers have been using the famous Alexa Fluor® dyes developed by Molecular Probes for everything from flow cytometry to oligonucleotide labeling. These sulfonated forms of common fluorophores (coumarin, rhodamine, fluorescein, and cyanine) are thought to be more stable, brighter, and less pH-sensitive than the native molecules (Ref: Panchuk-Voloshina et al. 1999). Antibody users are likely most familiar with Alexa Fluor® 488 and Alexa Fluor® 594 which when used together allow for the simultaneous staining of one protein in green and another in red and can be used in combination with the blue nuclear dye DAPI.
In our series of posts on different signaling pathways, let’s take a look today on Notch and its relevance in Acute Renal Failure (ARF).
A recent paper by Gupta et al. elucidated the role of the Notch pathway in kidney regeneration. This paper means an advance towards understanding potential therapeutic targeting of Notch signaling to enhance renal repair. Activation of the Notch pathway occurs following ARF. Pretreatment with the Notch ligand DLL4 enhanced recovery from ARF and represents a potential novel therapeutic option for regenerating the injured kidney.
However, compared to previous publications, as the authors mention in the paper, the use of different antibodies can affect the overall result of the experiment (as we all know!). In this specific case, Gupta et al. demonstrated increased expression of cleaved Notch1 and cleaved Notch2 as early as 1 h following reperfusion after 45 min of ischemia, and their findings are consistent with studies by Kobayashi et al. in a similar model of ARF with a few exceptions.
The paper by Kobayashi showed increased mRNA and protein expression of Delta-1, cleaved Notch2 only, while cleaved Notch1 was minimally detected under basal conditions or following injury. However, Gupta used the cleaved Notch-1 antibody from Rockland (see figure), and detected a robust signal for cleaved Notch1 with increased expression seen as early as 1 h following injury. These results were confirmed by immunohistochemistry using the Val1744 antibody. Therefore, both Notch1 and Notch2 are activated in the kidney following ARF.
Notch signaling has many roles, from neuronal function and development to the expansion of the hematopoietic stem cell compartment during bone development. Notch signaling pathways are a booming area of pharmacological research, due largely to the direct connection to human disease intervention.
Dengue Virus (DenV) is transmitted by mosquito vectors. It infects 50-100 million people each year and is at the origin of Dengue Fever and the more lethal Dengue Hemorrhagic Fever (DHF) and Shock Syndrome (DSS) leading to an estimated 500,000 cases of DHF and 22 000 deaths. The World Health Organization (WHO) estimates that 40% of the world’s population is at risk of infection.
In the June issue of Journal of Biomolecular Screening, investigators at San Diego State University (Dept of Biology) and Institute Pasteur Korea (Seoul, South Korea) developed a multiplexed cell-based assay for the identification of modulators of pre-membrane processing as a target for the discovery of DenV inhibitors. (1)
The DenV pre-membrane protein (prM) is an essential chaperone for the viral envelope protein which prevents premature fusion with vesicles during viral export. Inhibition of pre-membrane protein cleavage restricts fusion and represents, thus, a novel druggable target.
The new in vitro assay developed in this study, is the first described cell-based assay that monitors DenVprM processing within the classical secretory pathway. In a pilot screen of 1,280 small molecules on that assay, Thiostrepton, a known cyclopeptide Antibiotic and FOXM1 inhibitor, was identified as a novel positive hit in this assay (IC50=4.94 µM).
The utility of this novel assay has been proven by the identification of Thiostrepton (available at Focus Biomolecules cat. nr 10-2108) which may be a novel lead compound for the discovery of new drugs effective against Dengue Virus.
1- Stolp Z.D. et al. “A Multiplexed Cell-Based Assay for the Identification of Modulators of Pre-Membrane Processing as a Target against Dengue Virus” (2015) J. Biomol. Screen. 20:616-626. DOI: 10.1177/1087057115571247.
|Dengue Fever||Cat. Nr||Product Name||Application|
|Antibody for Dengue Virus||157MAB4043||Dengue virus Types 1,2,3,4 monoclonal antibody, clone BDI419||Dot,IF,EIA|
|Antibodies for Envelope Protein||157MAB8901||Dengue virus E-D3 monoclonal antibody, clone 5j122||ELISA,WB-Re|
|Antibodies for NS1 protein||157MAB13532||Dengue virus NS1 monoclonal antibody, clone HM026||ELISA,IF,LFIA|
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