The Reagent Roundup is made of brief summaries of publications in which researchers used Azure Biosystems reagents for Western blotting and Western blot quantitation in their studies. It is published every quarter. This quarter’s Reagent Roundup features publications from Duke University School of Medicine, University of Minho School of Medicine, the National Institutes of Health, and Boys Town National Research Hospital.
Phosphorylated MED1 links transcription recycling and cancer growth
Aberrant transcription goes hand-in-hand with oncogenesis. Chen et al1 used Chemi Blot Blocking Buffer and Radiance ECL in Western blot experiments investigating transcription recycling in cancer cells. Uncontrolled transcription initiation and elongation are known to be associated with tumor growth but the authors examined whether Pol II recycling, in which RNA polymerase II re-transcribes the same gene rather than being released after transcription is complete, is also associated with cancer. Using a recycling assay developed in their prior publications, the authors demonstrated that Mediator 1 (MED1), when phosphorylated by CDK9, drives Pol II recycling.
Phosphorylation of MED1 increased during prostate cancer progression and inhibiting CDK9 decreased MED1 phosphorylation, Pol II recycling, and prostate tumor growth. The results suggest MED1 phosphorylation and transcription recycling are involved in cancer growth, and MED1 phosphorylation may provide a biomarker to assess therapeutic response of cancers to CDK9 inhibitors.
Aripiprazole Offsets Mutant ATXN3-Induced Motor Dysfunction
Machado-Joseph disease (MJD) is a dominantly inherited progressive ataxia caused by expansion of a CAG repeat in the ataxin-3 gene. Jalles et al2 used Radiance ECL and AzureRed total protein stain, in addition to the Sapphire Biomolecular Imager, in a study investigating how the antipsychotic drug aripiprazole suppresses MJD pathogenesis. In a C elegans model of MJD, the authors found that aripiprazole improved motor performance and this improvement depended on dopamine D2-like and serotonin 5-HT1A and 5-HT2A receptors. Identifying the specific targets of aripiprazole may help develop new therapeutics for MJD with fewer side effects.
DISCOVER: Sapphire Biomolecular Imager
MARK2 regulates directed cell migration
During metastasis, cancer cells migrate by building out the cytoskeleton at the leading edge of the cell and retracting it at the rear. Pasapera et al used Azure’s Radiance ECL in a study of cancer cell cytoskeleton polarization. The authors investigated whether the kinase MARK2, known to regulate the microtubule cytoskeleton in other processes, plays a role in the polarization of the cytoskeleton and directed migration of cancer cells. In osteosarcoma cells, Western blot experiments demonstrated that MARK2 promotes stress fiber formation and myosin II activation and mediates inactivation of myosin phosphatase.
The data suggests MARK2 is a major regulator of cell contractility and adhesion that mediates cancer cell motility.
SHOP: Radiance ECL
Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli
Alport syndrome is a congenital, progressive glomerular disease that leads to the progressive loss of kidney function. Madison et al4 used Radiance ECL and TotalStain Q as well as an Azure 600 Imaging System in a study characterizing the glomerular basement membrane (GBM) in a mouse model of Alport syndrome. The investigators found that collagen a1(III) was deposited in the GBM of Alport mice; in wild type mice, collagen a1(III) is found only in the mesangium.
Quantitative Western blotting was carried out using total protein normalization with TotalStain Q staining as the control and the quantitative Westerns confirmed increased levels of collagen a1(III) in the glomeruli of Alport mice. The presence of collagen a1(III) was found to activate DDR1 receptors and lead to changes in gene expression consistent with podocyte injury. Lack of either of the two collagen receptors on podocytes has previously been shown to slow disease progression. The results indicate aberrant collagen-mediated co-receptor signaling through the DDR1 and a2b1 integrin receptors contribute to podocyte injury and renal pathology in Alport syndrome.
DISCOVER: Azure 600 Imaging System
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- Chen Z, Ye Z, Soccio RE, et al. Phosphorylated MED1 links transcription recycling and cancer growth. Nuc Acids Res. 2022;500(8):4450-4463.
- Jalles A, Vieira C, Pereira-Sousa J, et al. Aripiprazole offsets mutant ATXN3-induced motor dysfunction by targeting dopamine D2 and serotonin 1A and 2A receptors in elegans. Biomedicines. 2022;10(2):370.
- Pasapera AM, Heissler SM, Eto M, et al. MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization. Curr Biol. 2022;32(12):2704-2718.
- Madison J, Wilhelm K, Meehan DT, et al. Glomerular basement membrane deposition of collagen a1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin a2b J Pathol. 2022; doi: 10.1002/path.5969.