Use of short-lived green fluorescent protein for the detection of proteasome inhibition.
Human embryonic kidney (HEK293) cells were stably transduced with a retroviral vector containing an expression cassette for a short-lived green fluorescent protein (d2EGFP) and the neomycin resistance gene (Neor). When Neor HEK293 clones were treated with proteasome inhibitors, lactacystin or MG132, an increase in the constitutive levels of d2EGFP expression was observed. Based on flow cytometry, proteasome inhibitors induced a 5- to 10-fold increase in the fluorescent intensity of d2EGFP in HEK293 cell clones. However, in the presence of proteasome inhibitors, HEK293 clones showed a 4- to 6.5-fold increase in d2EGFP concentration as determined by western blot analysis. Our data suggest that d2EGFP is a useful indicator of proteasome inhibition. Therefore, stable expression of d2EGFP in mammalian cells is potentially useful for high-throughput screening of cDNAs or pharmaceutical drugs that repress proteasome functions in vivo.
Andreatta
C
C
University of Colorado Health Sciences Center, Denver, CO, USA.
Nahreini
P
P
Hovland
A R
AR
Kumar
B
B
Edwards-Prasad
J
J
Prasad
K N
KN
D002460:Cell Line; D003546:Cysteine Endopeptidases; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000032:analysis*; D046988:Proteasome Endopeptidase Complex; D012190:Retroviridae / Q000235:genetics*
D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex