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- Table of Contents
Facts about WD repeat domain-containing protein 83.
May act by recruiting signaling complexes to a particular upstream activator (By similarity). Also functions as a module in the assembly of a multicomponent scaffold for the ERK pathway, linking ERK answers to specific agonists.
Mouse | |
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Gene Name: | Wdr83 |
Uniprot: | Q9DAJ4 |
Entrez: | 67836 |
Belongs to: |
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WD repeat MORG1 family |
MAPK organizer 1; MGC4238; MORG1Mitogen-activated protein kinase organizer 1; WD repeat domain 83; WD repeat domain-containing protein 83
Mass (kDA):
34.444 kDA
Mouse | |
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Location: | 8|8 C3 |
Sequence: | 8; |
Ubiquitous.
The WDR83 marker can be used to study the role that the protein plays in HIF1A/HIF-1-alpha regulation. It is a negative regulator of HIF1A that promotes the degradation of this protein. It might recruit signaling compounds to upstream activators. It acts as a multi-component scaffold within the ERK pathway which links responses and agonists.
Many biological assays include the use of antibodies to Wdr83. They can be monoclonal or polyclonal and react with a variety of animal samples. Boster Bio's WDR83 antibodies are suitable for use in rabbit and mouse samples. The Wdr83 protein functions as a negative regulator of HIF1A, which promotes its degradation. It may also recruit signaling compounds to its upstream activator. It is a multicomponent scaffold of ERK pathways, linking ERK reactions to specific agonists.
The process of western blotting enables researchers to examine protein expression in cells or tissue homogenates. Western blotting can detect levels as low as picograms of protein. It is extremely sensitive. To improve reproducibility, Boster Bio has developed various Western blot detection methods, including multiplexing. This article will discuss each method as well as theoretical clarifications and troubleshooting ideas for widespread problems.
The membrane is the first step in western blotting. This membrane is available in two types: nitrocellulose and PVDF. The high affinity of nitrocellulose membranes to proteins makes them preferred, but they are difficult to reprob. PVDF membranes offer greater mechanical support but have a higher background. To minimize non-specific antibody binding, membranes must be blocked. In most cases, blocking involves using 5% BSA or nonfat dried milk.
Far-Western blotting is another method that can detect specific protein-protein interactions. The method involves electrophoresis, which separates the proteins into bands of a certain molecular weight. Secondary antibodies then identify these bands. These antibodies should produce a specific molecular band, which can then been determined with a protein staircase. Western blotting is becoming more complex as these methods become more sophisticated.
One of the biggest problems with western blot detection is that the IgG heavy chain is about 50 kDa and the IgG light chain is about 25 kDa. This makes it more difficult to identify the protein of concern. It also makes it harder to detect immunoprecipitated proteins with molecular weights over 50 kDa. This can be solved by switching to a different source for antigen.
Another method involves transfer of proteins to PVDF membranes using an ion-exchange technique. The proteins then transfer to nitrocellulose mA at 80 mA using buffer containing 5% BSA, 0.05% Tween-20 and 1 % Triton glycocine. After the membranes have been transferred the protein bands were detected by the ChemiDoc XRS Photo System from Biorad Laboratories.
We use a secondary antibody 1:600 in the anti protein SEMA4D (Bcl-2), Bax (Cleaved Caspase3) antibodies to detect proteins. These primary antibodies should then be diluted according to the manufacturer's protocol to a concentration of between 100-200 ng/ml. The results of the test should be recorded using a digital imager and luminometer. The second antibody should also be placed on top of the membrane.
There are many ways that proteins can be detected on a membrane. The AP enzyme uses different substrates and can get more information out of a membrane. Chemiluminescent is a detection method that uses an enzyme as a byproduct of a reaction to produce light. CCD imaging can also be fully automated, making it more sensitive than film-based detection methods. In addition, AP enzymes are available for quantitative analysis.
Inhibiting HIF-1a activity has been shown to block tumor growth in several studies. Hypoxia-related cancers such as those that benefit from hypoxia may find it advantageous to inhibit HIF-1a activity. It may also be necessary for malignant progress. Recent studies also suggest that hypoxia may enhance survival signals. These findings offer novel avenues for anticancer therapy. Anticancer therapies will likely target HIF-1a in near future based its OXPHOS status.
HIF-1a expression and translation is directly boosted by the PI3K/Akt/mTOR pathway. In normoxic conditions, aberrant stimulation of the Akt pathway activates HIF-1a. High ROS levels are associated with intermittent hypoxia and chemical toxins can activate HIF-1a. Finally, cancers expressing mutations in the TP53 tumor suppressor protein would also be suppressed.
It is not clear what role WDR83 plays in the downregulation HIF-1a. It is possible for WDR83 to block HIF-1a activation, but it depends on how the ERK pathway works. The protein interacts and can inhibit or enhance ERK activity. It may also promote HIF1A degradation.
For many years, research efforts have been ongoing to identify hypoxia-associated cancer cells. While there have been successful approaches, they are not applicable to all patients. They may not be effective across a wide range of patients. These limitations require that specific target genes be identified and the mechanisms of HIF1A -driven gene expression explored.
WDR83 negatively regulates other key genes in neurons, and inhibits HIF1A. This is particularly relevant for NK cell, where HIF1A represents a critical signaling pathway. Silenced APC/C also inhibits NMYC which is a hallmark for NB malignancy.
During hypoxia, tumor cells are inactivated by hypoxia-induced metabolites, which affect global DNA methylation. Tumor hypoxia reduces the activity the ten-eleven transcription enzyme (TETs). This indicates that hypoxia-induced global hypermethylation is an important effect of tumor hypoxia. Global DNA methylation is controlled by the gene WDR83.
This study was carried out in triplicates on HIF1A#B cells that had been silenced. We also used a non-silencing, lentiviral shRNAmir in order to knock down HIF1A within human cells. After 10 days of cells being treated with puromycin and RNA sequencing analysis, we found that WDR83 reduced the activity HIF1A/HIF-1A-alpha.
Researchers from several labs participated in this study. Researchers from the University of Toronto's Institute of Genetics discovered that WDR83 acts as a negative regulator of HIF1A/HIF-1A-alpha. These findings have implications in the development of cancer therapies. There is a need to investigate the mechanism by which WDR83 regulates tumor stemness and hypoxia promotes cell growth.
PMID: 15118098 by Vomastek T., et al. Modular construction of a signaling scaffold: MORG1 interacts with components of the ERK cascade and links ERK signaling to specific agonists.