HBZ promotes proteasomal degradation of c-Jun. A, HEK-293T
cells were transfected with 0.5 μg of pcDNA3-HA-c-Jun and 3 μg of either
pcDNA3 or pcDNA3-FLAG-HBZ. After 12 h, the cells were treated with or without
MG132 for 12 h. Cell lysates were immunoblotted with the antibodies indicated.
B, HEK-293T cells were transfected with 5 μg of pCAG-FLAG-HBZ or
control vector. After 36 h, the cells were treated with cycloheximide (50
μg/ml) and collected at the indicated times. Cell lysates were analyzed by
immunoblot analysis. C, upper panel: C8166 cells were
transfected using lentiviruses transcribing short hairpin RNAs against HBZ or
a nonspecific (n/s) sequence. The relative mRNA expression of HBZ,
c-Jun or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
evaluated by semiquantitative reverse transcription-PCR 7 days after
transfection. Efficiencies of lentivirus vector transfection, which were
determined by green fluorescent protein expression, were >95%. Lower
panel: C8166 cells were transfected as indicated in the upper
panel. After 7 days, the cells were treated with cycloheximide (50
μg/ml) and collected at the indicated times. Cell lysates were analyzed by
immunoblot analysis.