No transcriptional NF-κB activity after PrP infection

Copyright information:

Taken from "Alteration of NF-κB activity leads to mitochondrial apoptosis after infection with pathological prion protein"

Cellular Microbiology 2007;9(9):2202-2217.

Published online 15 Jun 2007

PMCID:PMC2048569.

© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd

A. Electromobility shift assay revealed NF-κB binding activity in PrP-infected neuroblastoma cells (Bos2). Nuclear extracts of Bos2 cells infected with RML6, treated with CD1 or stimulated with 10 ng ml TNF-α were incubated with P-labelled double-stranded oligonucleotide specific for NF-κB. The TNF-α sample was incubated with a 50-fold excess of cold NF-κB oligonucleotide as specific competitor or with a mutated oligonucleotide as control. B. κB precipitation experiment and detection using antibodies directed against p65, p50, p52 and Bcl-3. κB binding proteins were precipitated from nuclear extracts using an agarose-conjugated consensus κB oligonucleotide. The precipitates from either untreated controls, TNF-treated or RML6-infected Bos2 cells were divided and analysed in Western blots for the presence of the various NF-κB subunits. The immunoblot was quantified by scanning densitometry. The total signal intensities (p65 + p50, +p52 + Bcl-3) for the treated cells relative to the control cells and the portion of each subunits are given in percentage. C. Luciferase reporter gene assay showed no transcriptional NF-κB activity after PrP infection (0.2% or 0.02%), the value of relative light unit (RLU) per μg protein being equivalent for RML6- and CD1-treated cells. Mean values and SEM of three independent transfections are shown.