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. 2008 Dec 5;283(49):34273–34282. doi: 10.1074/jbc.M802527200

FIGURE 4.

FIGURE 4.

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.