Monoclonal validated for Post-Translational Modification studies

Knowing the Human genome better has allowed major advancements in Personalised Medicine. Nowadays, we can know (if we want) the likelihood to develop a given disease and/or how we will react to different pharmacological treatments. Examples of this include diseases like breast cancer (for diagnosis or estimation of likelihood) and lung cancer (for response to treatment), to name just a few.

That said, our genotype does not have the last word. Research in the last couple of decades has shown the power of other regulatory mechanisms, that may enhance or diminish the effect that our genotype will have on our health. Starting from basic healthy life styles, to other more subtle mechanisms, our genotype defines us, but not completely. Above genetics, we have epigenetics… and everything at the protein level. This post will focus on Post-Translational Modifications (PTM), because, after all, it’s the proteins that are the final effectors of a given response to a treatment or to an environmental stimulus.

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Ready-to-Use ELISAs to study Transcription Factors DNA binding

TFACT™ DNA-BINDING ELISA KITS » OCT2 TFACT™ DNA-BINDING ELISA

TFACT™ DNA-BINDING ELISA KITS » OCT2 TFACT™ DNA-BINDING ELISA by Assay Biotechnology. Source: tebu-bio.

Gene expression is regulated by different mechanisms. One of them is the binding of Transcription Factors (TF) to DNA sequences.

Traditionally, the study of TF-DNA interactions is made by several time-consuming and cumbersome: Electrophoretic Mobility Shift Assays (EMSA), Chromatin Immunoprecipitation, Western blotting, and expression of fused target and reporter genes.

ELISA-based formats now allow to have a more precise TF-DNA interaction study in addition to an ease of use.

These new tools significantly reduce the necessary runtime (within one day) and eliminate the need for harmful radioactive labeling. High sensitivity and signal-to-noise ratio are also guaranteed.

The TFact™ product-line belongs to these new ELISA-based Transcription Factor DNA binding assays. These indirect ELISAs allow an easy the detection and qualitative determination of the effects of phosphorylation on transcription factor activation profiles in a variety of nuclear and cell lysates from human, mouse and rat.

TFACT™ DNA-BINDING ELISA KITS » AML1 TFACT™ DNA-BINDING ELISA: AML1 (Phospho-Ser435)

The TFact™ AML1 DNA-Binding ELISA detects active AML1 in Hela Nuclear Extract. The Hela cells were grown 3 days in DMEM with 10% FBS and harvested for nuclear extract. The Hela cells were stimulated by PMA (200nM) before harvest.

TFACT™ DNA-BINDING ELISA Kits are available for various Transcription Factors and well-defined phosphorylated sequences: AML1, Jun, Androgen & Estogen Receptors, ATF2, CREB, FOXO, NFkB, STAT, p53, myb, SMAD, Sox …

Looking for simple but robust methods for studying TFs and the effect of phosphorylation in their activity?

Contact me for any further assistance!

Biomarkers and shear stress

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Costaining of pAkt-1S473 (red) and PECAM-1 (green) after 30 min flow-adaptation and 5 min ortho- (D) or retrograde flow (E). Image was obtained using Cat. No. 039200-301-J34L for p-Akt staining.

Research nowadays aims at working on models as similar as possible to the real physiological status. This includes the modification of cell culture conditions, For example, one should perform cell culture under “real” oxygen levels (e.g. hypoxia, normoxia, physioxia). For circulating cells, shear stress is a key factor, as cells behave in a different way depending on whether they are cultured under static or dynamic conditions. [Read more…]

Post-translational modifications regulate Ral GTPases

RalA and RalB GTPases regulate cell motility, morphology, signaling, vesicular trafficking, and endo/exocytosis. The regulation of these functions is critical for the development and spread of cancer, implicating Ral in oncogenesis and metastasis. Both isoforms are integral for Ras-mediated tumorigenesis, metastasis, and invasion. Despite sharing 82% amino acid sequence identity, effectors, and structural/biochemical properties, RalA and RalB have their own unique functions in oncogenesis due to distinct subcellular localization and differential effector interactions. Ral localization, binding partners, and function are regulated by post-translational modifications (PTMs).

print_logoIn their recent newsletter, Cytoskeleton Inc. summarize recent findings about the relevance of geranylgeranylation, carboxymethylation, palmitoylation, phosphorylation, and ubiquitination in regulating Ral activity, subcellular localization, effector binding, and ultimately, function.

You can download a copy of this newsletter, or if you have any questions or comments, don’t hesitate to get in contact through the form below.

Kits to measure RalA activation

If you’d like to get an overview about what’s available in the small G protein field, take a look at this Small GTPase product guide.

Phosphorylation of RhoA as a Signal Transduction Regulator

PTMs on Rho A

Post-translational Modifications of RhoA

Rho family GTPases are key regulators in a wide range of physiological processes, including cell motility, cell division, and neuronal development. Rho activity is regulated temporally and spatially by a variety of direct post-translational modifications (PTMs) that include prenylation, ubiquitination, oxidation, nitrosylation, and phosphorylation.

Cytoskeleton Inc. recently released a newsletter highlighting the control of RhoA function through phosphorylation. RhoA is a target for a growing number of kinases and as such, phosphorylation is emerging as a central theme in the regulation of this family of proteins (2).

The newsletter focussed on the mechanism of RhoA phosphorylation at Serine 188, which is mainly conducted by kinases like PKA and PKG (protein kinase A and protein kinase G) which are cyclic AMP-dependent and cyclic GMP-dependent respectively.

Furthermore, it looks at the physiological consequences of RhoA phosphorylation and future directions especially concerning the RhoA PTM involvement in diseases and potential  therapeutic options.

You can download a copy of this newsletter, or if you have any questions or comments, don’t hesitate to contact me through the form below.

Related to RhoA and PTM research:

References:

1. Stankiewicz T. & Linseman D. 2014. Rho family GTPases: key players in neuronal development, neuronal survival and neurodegeneration. Front. Cell. Neurosci. doi: 10.3389/fncel.2014.00314.
2. Boulter E. et al. 2012. Off the beaten paths: alternative and crosstalk regulation of Rho GTPases. FASEB J. 26, 469-479.

Tumour microenvironment – the kinome (II)

In a previous post, we discussed  kinome studies in the tumour microenvironment (TME). We described some solutions to study known markers, but we did not look at those cases in which the biomarkers associated to the kinome are unkown, and therefore some exploration is needed. [Read more…]

Tumour microenvironment and kinome studies

We all react to external factors, even the most cold-blooded person. We might hide our emotions, but there they are.

The same happens in cancer. Cells (both the tumour cells and the normal ones) react to the environment in one direction or another. In this post, we will see how “kinome” analysis might help Researchers in better understanding cellular interactions in tumour microenvironment (TME).

[Read more…]