Intracellular and extracellular PVA hydrolyzing enzymes are capable of degrading PVA and form molecules that were detected and examined in the cellular extract.
OGA was initially isolated from crude cellular extract, and it catalyzes hydrolytic cleavage of O-GlcNAc from proteins (Gao et al., 2001).
The filtered cellular extract was applied to BioFox 40 IDAlow (Knauer) resin charged with Ni2+ [1 column volume (CV) = 11 mL].
Luciferase activity was measured by liquid scintillation counting (Monolight 3010, PharMingen) using 20 µl of the cellular extract.
The bead pellet was mixed with 1 mg of protein of the cellular extract and mixed gently overnight.
As a source of recombinase activity, we used a cellular extract prepared from the D10 cell line [29] after heat-shock induced expression of core RAG1/2 proteins.
A similar result was obtained when the Alp14p-myc containing cellular extract was prepared from wild type cells (data not shown).
As an internal positive reference control, total cellular extract of DENV-3 infected cells was used as the capture antigen [24].
It is a minimal system in the sense, that only recPrP as substrate, buffer, and purified PrPSc as seed, but no cellular extract is needed.
MG-132 (1 µM) or 17-AAG (50 nM) were added to the cellular extract and incubated for 60 min. After adding 5 µl proteasome substrate II, the contents were incubated for additional 30 min at 37°C.
Whole cellular extracts from adult mouse brain, and from transiently and stably transfected N2A cells were prepared, and blotted on nitrocellulose after separation by 4 12% SDS-PAGE (50 µg of cellular extract per sample).
