HBZ promotes degradation of proteins that bind its bZIP domain but not
its N terminus. A, HBZ promotes the degradation of proteins that
bind its bZIP domain. Upper panel: HEK-293T cells were transfected
with pcDNA3-Myc-JunB, pcDNA3-HA-JunD, pCAG-FLAG-ATF1, pCAG-FLAG-ATF2,
pCAG-FLAG-ATF4, or pcDNA3-HA-CREB, with or without HBZ. Cell lysates were
immunoblotted with the appropriate epitope-tag antibodies. Lower
panel: HEK-293T cells were transfected with 2.5 μg of pcDNA3-FLAG-ATF2
together with 2.5 μg of pcDNA3-HA-HBZ or pcDNA3-HA-HBZ-ΔLZ, followed
by treatment with 20 μm MG132 for 12 h. Cell lysates were
immunoprecipitated with an anti-HA antibody, followed by immunoblotting with
an anti-FLAG antibody. Immunoblotting of whole cell lysates was performed
using the antibodies indicated. B, an interaction with HBZ is
responsible for target protein degradation. Upper panel: schematic
diagram of c-Jun and c-JunΔLZ used in this study. Middle panel:
HEK-293T cells were transfected as indicated and then treated with MG132 for
12 h. Cell lysates were immunoprecipitated with an anti-FLAG antibody and then
immunoblotted with an anti-HA antibody. LC indicates the
immunoglobulin light chain. Lower panel: HEK-293T cells were
transfected with pcDNA3-HA-c-JunΔLZ, with or without HBZ. Cell lysates
were immunoblotted with an anti-HA antibody. C, upper panel:
schematic diagram of p53 and c-JunLZ/p53. Middle panel: HEK-293T
cells were transfected as indicated and then treated with MG132 for 12 h. Cell
lysates were immunoprecipitated with an anti-FLAG antibody and then
immunoblotted with an anti-HA antibody. Lower panel: HEK-293T cells
were transfected with pcDNA3-FLAG-p53 or pcDNA3-FLAG-c-JunLZ/p53, with or
without HBZ. Cell lysates were immunoblotted with an anti-FLAG antibody.
D, HBZ does not promote degradation of proteins that bind its N
terminus. Upper panel: HEK-293T cells were transfected with
pcDNA3-HA-GADD34, pcDNA3-HA-MCM5, or pcDNA3-HA-MCM7, with or without HBZ. Cell
lysates were immunoblotted with an anti-HA antibody. Lower panel:
HEK-293T cells were transfected with 2.5 μg of pcDNA3-HA-GADD34,
pcDNA3-HA-MCM5, or pcDNA3-HA-MCM7, together with 2.5 μg of pcDNA3-FLAG-HBZ,
pcDNA3-FLAG-HBZ-N, or pcDNA3-FLAG-HBZ-ΔN60. Cell lysates were
immunoprecipitated with an anti-FLAG antibody and then immunoblotted with an
anti-HA antibody.