When the translated wild-type kinase was subjected to autophosphorylation conditions in the presence of Ca2+, CaM, Mg2+, and ATP, the Ca2+-independent activity (assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) increased from 5.8 +/- 0.7 to 26.5 +/- 2.1% of total activity (assayed in the presence of Ca2+/CaM).
To establish whether the improved stability of the I539T mutant was due to rescued conformation, we in vitro translated wild-type and ΔF508 NBD1 with or without suppressor mutations and monitored changes in proteolytic digestion (Figure 4A).
To find out whether ΔF508 misfolds NBD1 also in the absence of the other CFTR domains and out of biological context, we analyzed in vitro translated wild-type and ΔF508 NBdomainain in our assays and compared their protease susceptibilities.
The reaction was carried out at 37°C for 1 hr with the mixture including E1 (100 nM), E2 (5 µM), SUMO1 (50 µM), ATP (1 mM), and 35S-methionine-labeled in vitro-translated wild type FLAG-VHL or its mutants (2 µl) in total 20 µl volume.
In this protein degradation assay, 35S-labeled in vitro-translated wild-type BRCA1 and its mutants were subjected to cell extracts prepared from γ irradiation-treated cells.
Caspase-2 could cleave in vitro-translated wild-type Runx1, but relatively high concentrations of the enzyme were required to achieve this proteolysis.
In-vitro-translated wild-type human PPARα was generated by coupled in vitro transcription/translation (TNT system) using rabbit reticulocyte lysate as recommended by the supplier (Promega, Mannheim, Germany).
Indeed, in vitro kinase assays using purified recombinant cyclin/CDK complexes, revealed that both cyclin A-CDK2 and cyclin B-CDK1, but not cyclin E-CDK2, efficiently phosphorylated in vitro-translated wild-type (WT) FBXO28, but not a mutant protein where S344 was replaced with alanine (S344A) (Fig 2C).
We therefore initiated studies with HSA-hNEPv to investigate whether these findings translated to wild-type rats and non-human primates that have peripheral amyloid-β levels more similar to those found in patients with Alzheimer's disease.
DNA sequences were conceptually translated and aligned to wild type SIVmac239 in MacVector 9.0 trial version (Accelrys, Burlington, MA).
