The APEX DD-containing vectors encode proteins that have adjustable expression
at the protein level. The DD-fusion protein is transcriptionally under the
control of a CMV promoter (1). In the presence of Centry the protein is
stabilized, because Centry binds to the DD domain allowing the fusion protein to
accumulate in the cell in a dose-dependent manner (2). In the absence of Centry
or the removal of Centry from the media, the proteasome degrades the DD-fusion
Adjust protein expression by changing the media.
HEK293 cells were transfected with the DD-fused GFP. At the
beginning of the experiment, media with 500 nM Centry was added to the
cells to stabilize the GFP molecule. After 24 hours, the media was replaced
with standard growth media. Fluorescent micrographs were taken at the time
APEX System Delivers Dose-Dependent Protein Expression.
HEK293 cells were stably transfected with DD-fused GFP. Cells were treated
with increasing concentrations of APEX Centry at the beginning of the
experiment. APEX Centry was then removed after overnight incubation by
removing the media and adding fresh growth media. Fluorescent Activity is
expressed in arbitrary units and was monitored throughout the experiment
with the use of a fluorometer.
APEX Expression Constructs Yield Functional Proteins.
HEK293 cells were transiently transfected with Cignal™ KLF4
luciferase reporter (CCS-4036L) and the APEX
DD-fused KLF4. A renilla luciferase expression plasmid was used as a
transfection efficiency control. The media was changed after transfection to
either growth media or media containing 500 nM Centry. Cells were lysed after 24
hours and assayed for luciferase activity. Relative luciferase activity is shown
as the mean (± S.D.) of three independent experiments.
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