A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications

Mazet, Françoise; Dunster, Joanne L.; Jones, Chris I.; Vaiyapuri, Sakthivel; Tindall, Marcus J.; Fry, Michael; Gibbins, Jonathan M.

doi:10.1038/srep16995

Cited by 12 publications

(13 citation statements)

References 24 publications

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“…This method reliably identifies natural kinase substrates in cell samples. However, despite efforts to streamline the collection of quantitative temporal data (Bankston, Ku, & Feng, 2017;Mazet et al, 2015), this method is still limited in throughput and less amenable to accurate quantification.…”

Section: In-vitro Kinase Activity Assaysmentioning

confidence: 99%

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

Galicia

Aldehaiman

Hong

et al. 2019

Methods in Enzymology

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Protein tyrosine kinases (PTKs) are key signaling molecules and important drug targets. Although the efficient recombinant production of active PTKs is important for both pharmaceutical industry and academic research, most PTKs are still obtained from conventional, expensive and time-consuming insect-cell based expression. Host toxicity, kinase inactivity, insolubility and heterogeneity are among the reasons thought to preclude PTK expression in Escherichia coli. Herein we review these presumed roadblocks and their possible solutions for bacterial expression of PTKs, and give an overview on kinase activity assays. Finally, we report our experiences and observations with the kinases Src, Lyn and FAK as examples to illustrate implementation, effects and pitfalls of E. coli expression and in vitro assaying of PTKs.

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Section: In-vitro Kinase Activity Assaysmentioning

confidence: 99%

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

Galicia

Aldehaiman

Hong

et al. 2019

Methods in Enzymology

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“…When comparing the published protein copy numbers for proteins involved in the GPVI signalling pathway (GPVI, Syk, Cbl, and TULA‐2) in human and mouse platelets there are some striking differences (Figure D) . One difference of note is the 10‐fold increase in Syk molecules per mouse platelet compared to human platelets.…”

Section: Resultsmentioning

confidence: 99%

“…Human and mouse washed platelets (4 × 10 8 cells/mL) were prepared under non‐aggregating conditions with stirring using an AggRAM aggregometer (Helena Bioscience) for the indicated time points before lysis as described previously . Anti‐phospho Syk (Abcam; ab58575); anti‐phospho LAT (Abcam; ab68139); anti‐Actin antibody (C‐11; Santa Cruz; sc‐1615); anti‐Syk 4D10 (cell signalling).…”

Section: Methodsmentioning

confidence: 99%

“…Quantification of cell protein copy numbers is a critical step in the development of a predictive model of platelet activation . Quantitative proteomics, flow cytometry, and western blotting have been used to measure human platelet protein copy numbers . Most published reports of platelet protein copy numbers have been in humans and tend to focus on a single protein of interest.…”

Section: Introductionmentioning

confidence: 99%

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Interspecies differences in protein expression do not impact the spatiotemporal regulation of glycoprotein VI mediated activation

Dunster

Unsworth

Bye

et al. 2020

Journal of Thrombosis and Haemostasis

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Background: Accurate protein quantification is a vital prerequisite for generating meaningful predictions when using systems biology approaches, a method that is increasingly being used to unravel the complexities of subcellular interactions and as part of the drug discovery process. Quantitative proteomics, flow cytometry, and western blotting have been extensively used to define human platelet protein copy numbers, yet for mouse platelets, a model widely used for platelet research, evidence is largely limited to a single proteomic dataset in which the total amount of proteins was generally comparatively higher than those found in human platelets. Objectives:To investigate the functional implications of discrepancies between levels of mouse and human proteins in the glycoprotein VI (GPVI) signalling pathway using a systems pharmacology model of GPVI. Methods:The protein copy number of mouse platelet receptors was determined using flow cytometry. The Virtual Platelet, a mathematical model of GPVI signalling, was used to determine the consequences of protein copy number differences observed between human and mouse platelets. Results and conclusion:Despite the small size of mouse platelets compared to human platelets they possessed a greater density of surface receptors alongside a higher concentration of intracellular signalling proteins. Surprisingly the predicted temporal profile of Syk activity was similar in both species with predictions supported experimentally. Super resolution microscopy demonstrates that the spatial distribution of Syk is similar between species, suggesting that the spatial distribution of receptors and signalling molecules in activated platelets, rather than their copy number, is important for signalling pathway regulation. S U PP O RTI N G I N FO R M ATI O NAdditional supporting information may be found online in the Supporting Information section. How to cite this article: Dunster JL, Unsworth AJ, Bye AP, et al. Interspecies differences in protein expression do not impact the spatiotemporal regulation of glycoprotein VI mediated activation. J Thromb Haemost. 2020;18:485-496.

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“…Here we briefly describe recently established methods utilised for the measurement and quantification of protein copy numbers and their post translational modifications. Please see [ 70 ] for further details.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Early Steps of GPVI Signal Transduction by Phosphatases: A Systems Biology Approach

et al. 2015

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We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation.

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A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications

Cited by 12 publications

References 24 publications

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

Interspecies differences in protein expression do not impact the spatiotemporal regulation of glycoprotein VI mediated activation

Regulation of Early Steps of GPVI Signal Transduction by Phosphatases: A Systems Biology Approach

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