The cellular prion protein, PrPC, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrPSc is the causative agent of prion diseases. Conversion of PrPC into PrPSc is thought to take place at the cell surface or in endo-lysosomal organelles. Understanding the intracellular trafficking of PrPC may therefore help elucidating the conversion process. Here we describe a time-resolved fluorescence resonance energy transfer (FRET) assay reporting membrane expression and real-time internalization rates of PrPC. The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrPC trafficking. Simultaneous administration of FRET donor and acceptor anti-PrPC antibodies to living cells yielded a measure of PrPC surface density, whereas sequential addition of each antibody visualized the internalization rate of PrPC (Z'-factor > 0.5). RNA interference assays showed that suppression of AP2M1, RAB5A, VPS35 and M6PR blocked PrPC internalization, whereas downregulation of GIT2 and VPS28 increased it. PrPC cell surface expression was reduced by downregulation of RAB5A, VPS28 and VPS35, and enhanced by silencing EHD1. These data identify a network of proteins implicated in PrPC trafficking, and demonstrates the power of this assay for identifying modulators of PrPC trafficking.
from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2nlo836
via IFTTT
Εγγραφή σε:
Σχόλια ανάρτησης (Atom)
Δημοφιλείς αναρτήσεις
-
Publication date: Available online 4 January 2018 Source: European Journal of Radiology Author(s): Peiyao Zhang, Jing Wang, Qin Xu, Zhen...
-
Publication date: March 2017 Source: Free Radical Biology and Medicine, Volume 104 from #AlexandrosSfakianakis via Alexandros G.Sfak...
-
Dtsch med Wochenschr DOI: 10.1055/s-0043-100054 Hintergrund und Fragestellung Ein etablierter Weg, die optimale Behandlung von Tumorpatien...
-
Antibodies, Vol. 9, Pages 21: Construction of Ant... In Vivo and In Vitro Evaluation of Bull Semen Pro... Vertebral artery fenestration mimi...
-
Abstract Purpose F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) is emerging to be a useful tool in supporting the diag...
-
Does CBD Oil Lower Blood Pressure? This article was originally published at SundayScaries." Madeline Taylor POSTED ON January 13, 20...
-
Publication date: December 2017 Source: Advances in Biological Regulation, Volume 66 Author(s): Lauren Rusnak, Haian Fu The mitogen-activ...
-
Medicine by Alexandros G. Sfakianakis,Anapafseos 5 Agios Nikolaos 72100 Crete Greece,00302841026182, Butyric Acid from Probiotic Staphyloco...
-
Correction to: The IL-23p19/EBI3 heterodimeric cytokine termed IL-39 remains a theoretical cytokine in man The original article can be found...
Δεν υπάρχουν σχόλια:
Δημοσίευση σχολίου