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001-es BibID:BIBFORM072316
035-os BibID:(WoS)000425895600022 (Scopus)85041571964
Első szerző:Nagyné Szabó Ágnes Timea (vegyész)
Cím:The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes / Szabó Ágnes, Szendi-Szatmári Tímea, Ujlaky-Nagy László, Rádi Ildikó, Vereb György, Szöllősi János, Nagy Peter
Dátum:2018
ISSN:0006-3495
Megjegyzések:Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Megjelenés:Biophysical Journal. - 114 : 3 (2018), p. 688-700. -
További szerzők:Szendi-Szatmári Tímea (1989-) (molekuláris biológus) Ujlaky-Nagy László (1977-) (biofizikus) Rádi Ildikó Vereb György (1965-) (biofizikus, orvos) Szöllősi János (1953-) (biofizikus) Nagy Péter (1971-) (biofizikus)
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2.

001-es BibID:BIBFORM061401
Első szerző:Szöőr Árpád (orvos)
Cím:Cell confluence induces switching from proliferation to migratory signaling by site-selective phosphorylation of PDGF receptors on lipid raft platforms / Árpád Szöőr, László Ujlaky-Nagy, Gábor Tóth, János Szöllősi, György Vereb
Dátum:2016
ISSN:0898-6568
Megjegyzések:Platelet derived growth factor receptors (PDGFR) play an important role in tumor pathogenesis and are frequently overexpressed in glioblastoma. Earlier we have shown that only confluent glioblastoma cell cultures exhibit a biphasic calcium transient upon PDGF stimulation. Here, we examined how the change in cell density leads to differential cellular responses to the same PDGF stimulus. PDGF beta receptors and their specific phosphotyrosine residues were fluorescently colabeled on A172 and T98G glioblastoma cells. The distribution in cell membrane microdomains (lipid rafts) and the phosphorylation state of PDGFR was measured by confocal microscopy and quantitated by digital image processing. Corresponding bulk data were obtained by Western blotting. Activation of relevant downstream signaling pathways was assessed by immunofluorescence in confocal microscopy and by Western blot analysis. Functional outcomes were confirmed with bulk and single cell proliferation assays and motility measurements. In non-confluent (sparse) cultures PDGF-BB stimulation significantly increased phosphorylation of tyr716 specific for the Ras/MAPK pathway and tyr751 specific for the phosphoinositide 3-kinase/Akt pathway. As cell monolayers reached confluence, tyr771 and tyr1021 were the prominently phosphorylated residues. Tyr771 serves as adaptor for RasGAP, which inactivates the MAPK pathway, and tyr1021 feeds into the phospholipase Cgamma / PKC pathway. Coherent with this, MAPK phosphorylation, Ki67 positivity and proliferation dominated in dispersed cells, and could be abolished with inhibitors of the MAPK pathway. At the same time, RhoA activation, redistribution of cortactin to leading edges, and increased motility were the prominent output features in confluent cultures. Importantly, the stimulus-evoked confluence-specific changes in the phosphorylation of tyrosine residues occurred mainly in GM1-rich lipid microdomains (rafts). These observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs through a confluence dependent, lipid raft-based regulatory mechanism. In particular, cell division and survival in sparse cultures and inhibition of proliferation and promotion of migration in confluent monolayers. In our model, the ability to switch the final output of the same stimulus as a function of cell density could be a key to the balance of proliferation and invasion in malignant glioblastoma.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
lipid rafts
site-selective phosphorylation
quantitative confocal microscopy
contact inhibition
tumor proliferation
invasive tumor growth
cell migration
glioblastoma
PDGFR
Megjelenés:Cellular Signalling. - 28 : 2 (2016), p. 81-93. -
További szerzők:Ujlaky-Nagy László (1977-) (biofizikus) Tóth Gábor (1989-) (általános orvos) Szöllősi János (1953-) (biofizikus) Vereb György (1965-) (biofizikus, orvos)
Pályázati támogatás:K75752
OTKA
NK 101337
OTKA
Baross Gabor Program
Egyéb
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3.

001-es BibID:BIBFORM086412
035-os BibID:(absztrakt azonosító)C3-056P
Első szerző:Ujlaky-Nagy László (biofizikus)
Cím:Cell surface pattern of PDGF receptors affects signalling in glioblastoma cells / Ujlaky-Nagy László, Szöőr Árpád, Szöllősi János, Vereb György
Dátum:2005
ISSN:1742-464X
Tárgyszavak:Orvostudományok Klinikai orvostudományok idézhető absztrakt
folyóiratcikk
Megjelenés:Febs Journal. - 272 : Suppl1 (2005), p. 230. -
További szerzők:Szöőr Árpád (1984-) (orvos) Szöllősi János (1953-) (biofizikus) Vereb György (1965-) (absztraktok, könyvfejezetek)
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4.

001-es BibID:BIBFORM077483
Első szerző:Ujlaky-Nagy László (biofizikus)
Cím:Flow cytometric FRET analysis of protein interactions / László Ujlaky-Nagy, Péter Nagy, János Szöllősi, György Vereb
Dátum:2018
Megjegyzések:In the past decades, investigation of protein?protein interactions in situ in living or intact cells has gained expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments required con?rmation at the cellular level. Fo ? rster (?uorescence) resonance energy transfer (FRET)-based methods are excellent tools for determining proximity and supramolecular organization of biomolecules at the cell surface or inside the cell. This could well be the basis for the increasing popularity of FRET. In fact, the number of publications exploiting FRET has exploded since the turn of the millennium. Interestingly, most applications are microscope-based, and only a fraction employs ?ow cytometry, even though the latter offers great statistical power owed to the potentially huge number of individually measured cells. However, with the increased availability of multi-laser ?ow cytometers, strategies to obtain absolute FRET ef?ciencies can now be relatively facilely implemented. In this chapter, we intend to provide generally useable protocols for measuring FRET in ?ow cytometry. After a concise theoretical introduction, recipes are provided for successful labeling techniques and measurement approaches. The simple, quenching-based population-level measurement, the classic ratiometric, intensity-based technique providing cell-by-cell actual FRET ef?ciencies, and a more advanced version of the latter, allowing for cell-by-cell auto?uorescence correction are described. An Excel macro pre-loaded with spectral data of the most commonly used ?uorophores is also provided for easy calculation of average FRET ef?ciencies. Finally, points of caution are given to help design proper experiments and critically interpret the results.
Tárgyszavak:Orvostudományok Elméleti orvostudományok könyvfejezet
Förster resonance energy transfer
Fluorescence resonance energy transfer
Flow cytometry
Protein interactions
Molecular proximity
FCET
Megjelenés:Flow Cytometry Protocols / eds. Teresa S. Hawley, Robert G. Hawley. - p. 393-419. -
További szerzők:Nagy Péter (1971-) (biofizikus) Szöllősi János (1953-) (biofizikus) Vereb György (1965-) (biofizikus, orvos)
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5.

001-es BibID:BIBFORM002771
Első szerző:Vereb György (biofizikus, orvos)
Cím:Fluorescence correlation spectroscopy of living cells / Vereb G., Ujlaky-Nagy L., Friedländer E., Vámosi G., Szöllősi J.
Dátum:2007
Tárgyszavak:Természettudományok Biológiai tudományok könyvfejezet
könyvrészlet
Cells
Fluorescence
Microscopy
Research
Megjelenés:Modern research and educational topics in microscopy / ed. Mendez-Vilas, A., Labajos-Broncano, L. - p. 848-854.
További szerzők:Ujlaky-Nagy László (1977-) (biofizikus) Friedländer Elza (1980-) (biofizikus) Vámosi György (1967-) (biofizikus) Szöllősi János (1953-) (biofizikus)
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