Western Blotting Reagents Roundup – August 2023

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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. This quarter, we’re highlighting four recent publications that used Azure reagents to achieve excellent Western blotting results.

Featured Studies in this Reagent Roundup

AzureSpectra secondary antibodies and AzureRed Protein Stain used in a study of the effects of JAK-STAT inhibitors on thrombosis risk

Inhibition of TNF-alpha-mediated STAT1 and STAT3 with ruxolitinib and fedratinib. AzureSpectra-conjugated secondary antibodies were used to detect primary antibodies
Figure 1 from Beckman et al (2023). Inhibition of TNF-alpha-mediated STAT1 and STAT3 with ruxolitinib and fedratinib. Licensed under CC BY 4.0. AzureSpectra-conjugated secondary antibodies were used to detect primary antibodies. Western blots (Panel A) were imaged using the Azure c600 imager.

Activation of vascular endothelial cells occurs in a range of pathologic states including COVID-19 and myeloproliferative neoplasms (MPNs). The JAK-STAT signal transduction pathway is a key regulator of proinflammatory signaling. Mutations in JAK can allow ligand-independent signaling which is associated with vascular activation and increased risk of thrombosis. JAK-STAT inhibitors are being studied as potential treatments of inflammatory conditions including MPNs, COVID-19, rheumatoid arthritis, and more. However, some clinical data suggests that JAK-STAT inhibitors could increase thrombosis risk.

In recent work, Beckman et al investigated the effects of JAK-STAT inhibitors ruxolitinib and fedratinib on pro-thrombotic and pro-inflammatory signaling in endothelial cells. A variety of approaches were used to assess multiple markers of endothelial activation and cell adhesion. In one series of experiments, fluorescent Western blots were conducted to measure levels of proteins in the signaling pathway. AzureSpectra secondary antibodies labeled with visible and NIR fluorescent dyes were used for detection, and the blots were imaged on an Azure c600 imager. In addition, the blots were stained with AzureRed Protein Stain before blocking to check protein transfer. The results of the study indicate that JAK-STAT inhibitors may reduce the production of pro-inflammatory and pro-adhesive factors in endothelial cells in response to TNF-alpha stimulation.

Since the release of this publication, the c600 Imaging System has been succeeded by the new Azure 600 Imaging System. This upgraded systems is a high-performance instrument capable of NIR fluorescence, visible fluorescence, and chemiluminescence.

Study demonstrating the effectiveness of targeted pseudouridinylation to bypass premature stop codons in disease causing mutations

Several genetic diseases are caused by point mutations that change a sense codon into a stop codon. These nonsense mutations result in stop codons that cause translation to stop prematurely such that full-length proteins are not made. Premature stop codons also cause the mRNA to be degraded via nonsense-mediated mRNA decay.

In a recent publication, Adachi et al applied a strategy that they previously developed in yeast to remove the premature stop codon from a disease-causing protein in cultured human cells. Guide RNAs were used to direct the targeted conversion of the uridine in the premature stop codon into a pseudouridine. The resulting codon is no longer read as a stop codon, and the full-length protein is translated. The authors ran chemiluminescent Western blots to assess protein expression in the presence and absence of the guide RNAs. The Westerns were activated using Radiance Plus substrate and imaged on an Azure c300 imaging system. The results confirmed that targeted pseudouridylation successfully suppressed nonsense-mediated mRNA decay and promoted premature stop codon readthrough in a disease model.

Since the release of this publication, the c300 Imaging System has been succeeded by the new Azure 300 Imaging System. It offers the simplicity, speed and sensitivity of film detection, with better resolution and more quantitative results.

Total protein stain used in a study characterizing the functional consequences of a disease-causing mutation in a protein required for mitochondrial fusion

Mutations in Mfn2, a protein required for mitochondrial outer membrane fusion, cause CMT2A, Charcot-Marie-Tooth Disease Type 2, an inherited sensory motor neuropathy. 

In recent work, Sloat and Hoppins characterized the disease-causing mutation Mfn2-S350P. It is hypothesized that a large conformational change in the Hinge 2 domain of Mfn2 is important for membrane fusion. To investigate this, the authors expressed the mutant protein (and an analogous mutation in a related protein, Mfn1) in mouse cells. Abnormal clustering of mitochondria was observed. To confirm that the mitochondrial clustering was not due to altered microtubule transport, the authors knocked down expression of the dynein heavy chain protein using shRNA. Quantitative chemiluminescent Western blots activated with Radiance Plus and imaged on an Azure Sapphire Biomolecular Imager were used to confirm the knockdown.

The protein levels were quantified using a total protein stain from Azure, normalizing the signal of interest to that of total protein. The data indicate that the mutant proteins induce perinuclear clusters via mitochondrial tethering that is not dependent on dynein-mediated transport and support a model in which conformational change at the Hinge 2 domain is required to progress from tethering to membrane fusion.

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

Both Radiance and Radiance Q used in a study investigating whether the protein TSPO/PBR is required for steroidogenesis

Western blot showing VDAC-1 protein levels in various tissues visualized with Radiance ECL from Azure Biosystems.
Figure 8 from Liere et al (2023). Assessing VDAC-1 protein levels in various tissues using Western blot analysis. Licensed under CC BY 4.0. Chemiluminescence using Azure ECL Radiance or Radiance Q was used to visualize protein bands.

The protein TSPO/PBR has been thought to be required for mitochondrial cholesterol transport and therefore essential for steroid production. In their recent study, Liere et al examined the steroid profile across multiple tissues of TSPO/PBR knockout mice to determine if and how steroidogenesis depends on this protein. TSPO/PBR is highly conserved and is expressed ubiquitously, including in tissues that synthesize steroid hormones.

Prior characterization of TSPO/PBR knockout mice has focused on a small number of steroids and has not definitively answered the question of the role of TSPO/PBR in steroid synthesis.

In the present work, the authors sought to comprehensively analyze the steroid profiles of the brain, adrenal glands, testes, and plasma of male knockout mice using GC-MS/MS, a method of gas chromatography followed by mass spectrophotometry. In addition, the researchers conducted chemiluminescent Western blots to measure levels of proteins that might functionally associate with TSPO/PBR. The Westerns were activated using Azure’s Radiance and Radiance Q chemiluminescent substrates. The data revealed that TSPO/PBR has only a limited and indirect effect on steroidogenesis. The levels of proteins examined by Western blot and the levels of the majority of steroids assessed did not differ between wild type and knockout mice. The authors propose that the molecular function of TSPO/PBR requires further study.

Find more publications using Azure reagents and imaging systems on our publications list, or contact us directly for assistance with a specific product by using the form below.

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SOURCES

  1. Beckman JD, DaSilva A, Aronovich E, et al. JAK-STAT inhibition reduces endothelial prothrombotic activation and leukocyte–endothelial proadhesive interactions. J Thromb Haemost. 2023; S1538-7836(23)00081-8.
  2. Adachi H, Pan Y, He X, et al. Targeted pseudouridylation: An approach for suppressing nonsense mutations in disease genes. Mol Cell. 2023;83:637-651.
  3. Sloat SR, Hoppins S. A dominant negative mitofusin causes mitochondrial perinuclear clusters because of aberrant tethering. Life Sci Alliance. 2023;6(1):e202101305.
  4. Liere P, Liu GJ, Pianos A, et al. The Comprehensive steroidome in complete TSPO/PBR knockout mice under basal conditions. Int J Mol Sci. 2023;24(3):2474.

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