Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin.
AuthorsSoosairajah J, Maiti S, Wiggan O, Sarmiere P, Moussi N, Sarcevic B, Sampath R, Bamburg JR, Bernard O
JournalEMBO J
PubMed ID15660133
'Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the ... More
Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis.
AuthorsChen Z, Southwick K, Thulin CD
JournalJ Biomol Tech
PubMed ID15585821
'Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary ... More
Critical role of serine 465 in isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3.
AuthorsHuang Y, Feng X, Sando JJ, Zuo Z
JournalJ Biol Chem
PubMed ID17062570
'Glutamate transporters (also called excitatory amino acid transporters, EAATs) bind extracellular glutamate and transport it to intracellular space to regulate glutamate neurotransmission and to maintain extracellular glutamate concentrations below neurotoxic levels. We previously showed that isoflurane, a commonly used anesthetic, enhanced the activity of EAAT3, a major neuronal EAAT. This ... More
Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program.
AuthorsTannu NS, Rao VK, Chaudhary RM, Giorgianni F, Saeed AE, Gao Y, Raghow R
JournalMol Cell Proteomics
PubMed ID15286212
'When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and ... More
Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry.
'Protein phosphorylation plays a vital role in the regulation of most aspects of cellular activity, being key to propagating messages within signal transduction pathways and to modulating protein function. Pro-Q Diamond phosphoprotein gel stain is suitable for the fluorescence detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl ... More
A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.
AuthorsGarcía A, Senis YA, Antrobus R, Hughes CE, Dwek RA, Watson SP, Zitzmann N
JournalProteomics
PubMed ID16941570
'Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for ... More
A sensitive method for determining the phosphorylation status of natriuretic peptide receptors: cGK-Ialpha does not regulate NPR-A.
AuthorsBryan PM, Smirnov D, Smolenski A, Feil S, Feil R, Hofmann F, Lohmann S, Potter LR
JournalBiochemistry
PubMed ID16430226
'Natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) are transmembrane guanylyl cyclases that catalyze the synthesis of cGMP in response to natriuretic peptides. Phosphorylation and dephosphorylation regulate these receptors and have been traditionally studied by (32)PO(4) labeling of transfected cells. However, this approach cannot be used to ... More
Two-dimensional gel proteome reference map of blood monocytes.
AuthorsJin M, Diaz PT, Bourgeois T, Eng C, Marsh CB, Wu HM
JournalProteome Sci
PubMed ID16948843
'BACKGROUND: Blood monocytes play a central role in regulating host inflammatory processes through chemotaxis, phagocytosis, and cytokine production. However, the molecular details underlying these diverse functions are not completely understood. Understanding the proteomes of blood monocytes will provide new insights into their biological role in health and diseases. RESULTS: In ... More
'Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, causes human monocyte/macrophage-tropic ehrlichiosis. In this study, proteomic approaches were used to demonstrate host cell-specific antigenic expression by E. chaffeensis. The differentially expressed antigens include those from the 28-kDa outer membrane protein (p28-Omp) multigene locus. The proteins expressed in infected macrophages are the products ... More
Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics.
AuthorsGohla A, Birkenfeld J, Bokoch GM
JournalNat Cell Biol
PubMed ID15580268
Cofilin is a key regulator of actin cytoskeletal dynamics whose activity is controlled by phosphorylation of a single serine residue. We report the biochemical isolation of chronophin (CIN), a unique cofilin-activating phosphatase of the haloacid dehalogenase (HAD) superfamily. CIN directly dephosphorylates cofilin with high specificity and colocalizes with cofilin in ... More
A two-dimensional electrophoresis map of Chinese hamster ovary cell proteins based on fluorescence staining.
AuthorsHayduk EJ, Choe LH, Lee KH
JournalElectrophoresis
PubMed ID15300775
We report on the development of a detailed two-dimensional electrophoresis map of Chinese hamster ovary (CHO) cell proteins based on fluorescence staining and tandem time-of-flight (TOF/TOF)-mass spectrometry. We observed a 71% success rate in the identification of proteins even though the CHO genome is not sequenced. The map consists of ... More
Determination of in vivo protein phosphorylation in photosynthetic membranes.
AuthorsVainonen JP, Vener AV, Aro EM,
JournalMethods Mol Biol
PubMed ID19083170
Light- and redox-controlled reversible phosphorylation of thylakoid proteins regulates short- and long-term acclimation of plants to environmental cues. The major phosphoproteins in thylakoids belong to photosystem II and its light-harvesting antenna but phosphorylation of subunits of other thylakoid protein complexes has been detected as well. The detection methods include electrophoretic ... More
A high-resolution two dimensional Gel- and Pro-Q DPS-based proteomics workflow for phosphoprotein identification and quantitative profiling.
AuthorsAgrawal GK, Thelen JJ,
JournalMethods Mol Biol
PubMed ID19241001
The two-dimensional (2-D) gel-based proteomics platform remains the workhorse for proteomics and is fueled by a number of key improvements, including fluorescence-based stains for detection and quantification of proteins and phosphoproteins with high sensitivity and linear dynamic ranges. One such stain is Pro-Q diamond phosphoprotein stain (Pro-Q DPS), which binds ... More
Detection of post-translational modifications by fluorescent staining of two-dimensional gels.
AuthorsJacob AM, Turck CW,
JournalMethods Mol Biol
PubMed ID18373247
Post-translational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial in the discovery of biomarkers of disease. Recent commercial availability of fluorescent dyes specifically staining PTMs of proteins such as phosphorylation and glycosylation enables the specific ... More
Structural and Functional Characterization of NikO, an Enolpyruvyl Transferase Essential in Nikkomycin Biosynthesis.
AuthorsOberdorfer G, Binter A, Ginj C, Macheroux P, Gruber K,
JournalJ Biol Chem
PubMed ID22810238
Nikkomycins are peptide-nucleoside compounds with fungicidal, acaricidal, and insecticidal properties because of their strong inhibition of chitin synthase. Thus, they are potential antibiotics especially for the treatment of immunosuppressed patients, for those undergoing chemotherapy, or after organ transplants. Although their chemical structure has been known for more than 30 years, ... More
Protein gel staining methods: an introduction and overview.
AuthorsSteinberg TH,
JournalMethods Enzymol
PubMed ID19892191
Laboratory scientists who encounter protein biochemistry in many of its myriad forms must often ask: is my protein pure? The most frequent response: run a denaturing SDS polyacrylamide gel. Running this gel raises another series of considerations regarding detection, quantitation, and characterization and so the next questions invariably center on ... More
Increased phosphorylation of tropomyosin, troponin I, and myosin light chain-2 after stretch in rabbit ventricular myocardium under physiological conditions.
AuthorsMonasky MM, Biesiadecki BJ, Janssen PM,
JournalJ Mol Cell Cardiol
PubMed ID20298699
After a change in muscle length, there is an immediate intrinsic response in the amount of developed force, followed by a slower response. Although it has been well documented that the slow force response is at least in part generated by modification of calcium handling, it is unclear whether regulation ... More
A universal assay for vitellogenin in fish mucus and plasma.
Expression of vitellogenin (VTG) in male fish has become a widely used biomarker of exposure to environmental estrogens. Vitellogenin is usually measured in blood by immunoassays that require species-specific antibodies. In this paper, we describe a universal assay that is based on the high-molecular weight and extensive phosphoserine content of ... More
Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining.
AuthorsStasyk T, Morandell S, Bakry R, Feuerstein I, Huck CW, Stecher G, Bonn GK, Huber LA
JournalElectrophoresis
PubMed ID15966015
Here we combine a standard two-dimensional difference gel electrophoresis (DIGE) protocol with subsequent post-staining of gels with phosphospecific fluorescent Pro-Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2-DE-gels. We established this protocol within a functional proteomics experiment. Mammary ... More
A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia.
AuthorsNagata-Ohashi K, Ohta Y, Goto K, Chiba S, Mori R, Nishita M, Ohashi K, Kousaka K, Iwamatsu A, Niwa R, Uemura T, Mizuno K
JournalJ Cell Biol
PubMed ID15159416
Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, ... More
Phosphoprotein analysis: from proteins to proteomes.
AuthorsDelom F, Chevet E
JournalProteome Sci
PubMed ID16854217
Characterization of protein modification by phosphorylation is one of the major tasks that have to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification occurring mainly on serine, threonine and tyrosine residues that can regulate enzymatic activity, subcellular localization, complex formation and degradation of proteins. The understanding ... More
Use of a fluorescent phosphoprotein dye to characterize oxidative stress-induced signaling pathway components in macrophage and epithelial cultures exposed to diesel exhaust particle chemicals.
AuthorsWang M, Xiao GG, Li N, Xie Y, Loo JA, Nel AE
JournalElectrophoresis
PubMed ID15880549
A large body of evidence has shown that exposure to ambient particulate matter (PM) leads to asthma exacerbation through an excitation of allergic inflammation. Utilizing diesel exhaust particles (DEPs) as a model air pollutant, we and others have demonstrated that PM contains redox-active chemicals that generate inflammation through an oxidative ... More
Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain.
AuthorsMurray J, Marusich MF, Capaldi RA, Aggeler R
JournalElectrophoresis
PubMed ID15300771
We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained ... More
Proteome and phosphoproteome dynamic change during cell dedifferentiation in Arabidopsis.
AuthorsChitteti BR, Peng Z
JournalProteomics
PubMed ID17407188
Cell dedifferentiation is a cell fate switching process in which a differentiated cell reverts to a status with competence for cell division and organ regeneration like an embryonic stem cell. Although the phenomenon of cell dedifferentiation has been known for over two and a half centuries in plants, little is ... More
Calcium signal-induced cofilin dephosphorylation is mediated by Slingshot via calcineurin.
AuthorsWang Y, Shibasaki F, Mizuno K
JournalJ Biol Chem
PubMed ID15671020
Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca2+ concentrations, signaling mechanisms mediating Ca2+-induced cofilin dephosphorylation have remained unknown. We investigated the role of Slingshot (SSH) 1L, a ... More
Combining microscale solution-phase isoelectric focusing with Multiplexed Proteomics dye staining to analyze protein post-translational modifications.
AuthorsSchulenberg B, Patton WF
JournalElectrophoresis
PubMed ID15300774
Previously, a strategy for rapidly identifying mitochondrial phosphoproteins was presented that involves prefractionating multisubunit complexes by sucrose gradient centrifugation, followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and selective staining of phosphoproteins and total protein with fluorescent dyes [1]. Though suitable for evaluating the mitochondrial proteome, which consists of numerous ... More
Differential phosphoprotein mapping in cancer cells using protein microarrays produced from 2-D liquid fractionation.
AuthorsPal M, Moffa A, Sreekumar A, Ethier SP, Barder TJ, Chinnaiyan A, Lubman DM
JournalAnal Chem
PubMed ID16448042
A combination of protein microarrays and two-dimensional liquid-phase separation of proteins has been used for global profiling of the phosphoproteome in human breast cancer cells. This method has been applied to study changes in phosphorylation profile resulting from treatment of the cancer cells with PD173074, a known receptor tyrosine kinase ... More
Blockade of class IA phosphoinositide 3-kinase in neutrophils prevents NADPH oxidase activation- and adhesion-dependent inflammation.
AuthorsGao XP, Zhu X, Fu J, Liu Q, Frey RS, Malik AB
JournalJ Biol Chem
PubMed ID17197441
We examined the role of class IA phosphoinositide 3-kinase (PI3K) in the regulation of activation of NADPH oxidase in PMNs and the mechanism of PMN-dependent lung inflammation and microvessel injury induced by the pro-inflammatory cytokine TNF-alpha. TNF-alpha stimulation of PMNs resulted in superoxide production that was dependent on CD11b/CD18-mediated PMN ... More
Phosphoproteomic analysis of neuronal cell death by glutamate-induced oxidative stress.
AuthorsKang TH, Bae KH, Yu MJ, Kim WK, Hwang HR, Jung H, Lee PY, Kang S, Yoon TS, Park SG, Ryu SE, Lee SC
JournalProteomics
PubMed ID17610204
Oxidative stress is one of the major causes of neuronal cell death in disorders such as perinatal hypoxia and ischemia. Protein phosphorylation is the most significant PTM of proteins and plays an important role in stress-induced signal transduction. Thus, the analysis of alternative protein phosphorylation states which occur during oxidative ... More
The nerve hemoglobin of the bivalve mollusc Spisula solidissima: molecular cloning, ligand binding studies, and phylogenetic analysis.
AuthorsDewilde S, Ebner B, Vinck E, Gilany K, Hankeln T, Burmester T, Kreiling J, Reinisch C, Vanfleteren JR, Kiger L, Marden MC, Hundahl C, Fago A, Van Doorslaer S, Moens L
JournalJ Biol Chem
PubMed ID16352603
Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (microM). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme ... More
Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice.
AuthorsChiba Y, Ueno A, Shinozaki K, Takeyama H, Nakazawa S, Sakai H, Misawa M
JournalRespir Res
PubMed ID15638941
BACKGROUND: It has recently been suggested that RhoA plays an important role in the enhancement of the Ca2+ sensitization of smooth muscle contraction. In the present study, a participation of RhoA-mediated Ca2+ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined. ... More
Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye.
The phosphorylation of mitochondrial proteins is pivotal to the regulation of respiratory activity in the cell and to signaling pathways leading to apoptosis, as well as for other vital mitochondrial processes. A number of protein kinases have been identified in mitochondria but the physiological substrates for many of these remain ... More
Functional effects of rho-kinase-dependent phosphorylation of specific sites on cardiac troponin.
AuthorsVahebi S, Kobayashi T, Warren CM, de Tombe PP, Solaro RJ
JournalCirc Res
PubMed ID15774859
We tested the hypothesis that activation of Rho-A-dependent kinase (ROCK-II) alters cardiac myofilament response to Ca2+ by mechanisms involving phosphorylation of thin filament proteins. We determined effects of a constitutively active form of ROCK-II on ATPase activity and tension development in detergent-extracted (skinned) fiber bundles isolated from mouse left ventricular ... More
Proteomic analysis of in vivo phosphorylated synaptic proteins.
AuthorsCollins MO, Yu L, Coba MP, Husi H, Campuzano I, Blackstock WP, Choudhary JS, Grant SG
JournalJ Biol Chem
PubMed ID15572359
In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue ... More
Toward functional proteomics of alveolar macrophages.
AuthorsWu HM, Jin M, Marsh CB
JournalAm J Physiol Lung Cell Mol Physiol
PubMed ID15757951
Alveolar macrophages (AM) belong to a phenotype of macrophages with distinct biological functions and important pathophysiological roles in lung health and disease. The molecular details determining AM differentiation from blood monocytes and AM roles in lung homeostasis are largely unknown. With the use of different technological platforms, advances in the ... More
Mitochondrial matrix phosphoproteome: effect of extra mitochondrial calcium.
AuthorsHopper RK, Carroll S, Aponte AM, Johnson DT, French S, Shen RF, Witzmann FA, Harris RA, Balaban RS
JournalBiochemistry
PubMed ID16489745
Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondrion-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondrial matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions ... More
Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase.
AuthorsWang X, Goshe MB, Soderblom EJ, Phinney BS, Kuchar JA, Li J, Asami T, Yoshida S, Huber SC, Clouse SD
JournalPlant Cell
PubMed ID15894717
Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in ... More
Functional analyses of placental protein 13/galectin-13.
AuthorsThan NG, Pick E, Bellyei S, Szigeti A, Burger O, Berente Z, Janaky T, Boronkai A, Kliman H, Meiri H, Bohn H, Than GN, Sumegi B
JournalEur J Biochem
PubMed ID15009185
Placental protein 13 (PP13) was cloned from human term placenta. As sequence analyses, alignments and computational modelling showed its conserved structural and functional homology to members of the galectin family, the protein was designated galectin-13. Similar to human eosinophil Charcot-Leyden crystal protein/galectin-10 but not other galectins, its weak lysophospholipase activity ... More
Proteomic analysis of rat liver phosphoproteins after treatment with protein kinase inhibitor H89 (N-(2-[p-bromocinnamylamino-]ethyl)-5-isoquinolinesulfonamide).
AuthorsDavis MA, Hinerfeld D, Joseph S, Hui YH, Huang NH, Leszyk J, Rutherford-Bethard J, Tam SW
JournalJ Pharmacol Exp Ther
PubMed ID16687476
Therapeutic strategies focused on kinase inhibition rely heavily on surrogate measures of kinase inhibition obtained from in vitro assay systems. There is a need to develop methodology that will facilitate measurement of kinase inhibitor activity or specificity in tissue samples from whole animals treated with these compounds. Many of the ... More
Functional characterization of two-dimensional gel-separated proteins using sequential staining.
Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications ... More
Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis.
AuthorsAllanore Y, Borderie D, Périanin A, Lemaréchal H, Ekindjian OG, Kahan A
JournalArthritis Res Ther
PubMed ID15642147
We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O2*-) production by peripheral ... More
Contact inhibition of hepatocyte growth regulated by functional association of the c-Met/hepatocyte growth factor receptor and LAR protein-tyrosine phosphatase.
AuthorsMachide M, Hashigasako A, Matsumoto K, Nakamura T
JournalJ Biol Chem
PubMed ID16415345
Contact inhibition, the inhibition of cell proliferation by tight cell-cell contact is a fundamental characteristic of normal cells. Using primary cultured hepatocytes, we investigated the mechanisms of contact inhibition that decrease the mitogenic activity of hepatocyte growth factor (HGF), focusing on the regulation of c-Met/HGF-receptor activation. In hepatocytes cultured at ... More
Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye.
Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be ... More
Use of proteomics to demonstrate a hierarchical oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line.
AuthorsXiao GG, Wang M, Li N, Loo JA, Nel AE
JournalJ Biol Chem
PubMed ID14522998
Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM) and cardiorespiratory morbidity and mortality. Although the biological mechanisms of these adverse effects are unknown, emerging data suggest a key role for oxidative stress. Ambient PM and diesel exhaust particles (DEP) contain redox cycling organic chemicals ... More
Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye.
Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of ... More
Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore.
A new formulation of the small-molecule organic fluorophore, Pro-Q Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins directly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to membranes, stained and destained, in an analogous manner ... More
Frequency-dependent acceleration of relaxation involves decreased myofilament calcium sensitivity.
AuthorsVarian KD, Janssen PM
JournalAm J Physiol Heart Circ Physiol
PubMed ID17209002
The force-frequency relationship is an intrinsic modulator of cardiac contractility and relaxation. Force of contraction increases with frequency, while simultaneously a frequency-dependent acceleration of relaxation occurs. While frequency dependency of calcium handling and sarcoplasmic reticulum calcium load have been well described, it remains unknown whether frequency-dependent changes in myofilament calcium ... More