sHBZ promotes p65 degradation through a ubiquitination-dependent pathway. (A) sHBZ repressed the level of p65 in a dose-dependent manner. 293FT cells were transfected with 1 μg pEF-p65 and various amounts of mycHis-sHBZ (0.2, 0.5, 1, 2, and 5 μg). After 36 hours, the cell lysates were subjected to immunoblotting. (B) Activation and leucine-zipper domains of sHBZ were necessary for suppression of p65. 293FT cells were transfected with 50 ng pEF-p65 and 250 ng either mycHis-sHBZ or its mutants. At 36 hours after transfection, the level of p65 mRNA was analyzed by semiquantitative RT-PCR. The levels of GAPDH mRNA are shown as internal control (top panel). Whole cell lysates were subjected to immunoblotting (bottom panel). (C) sHBZ-mediated suppression of p65 protein is caspase independent. Top panel: 293FT cells were transfected with FLAG-p65 together with mycHis-sHBZ. The caspase inhibitor z-VAD-fmk was added 2 hours before transfection. At 48 hours after transfection, cell lysates were subjected to immunoblotting. Bottom panel: Jurkat cells were cultured in the presence of indicated drugs for 24 hours. Cell death was analyzed by annexin V staining. (D,E) sHBZ accelerated the ubiquitination of p65. 293FT cells were transfected with FLAG-p65, HA-ubiquitin, and either mycHis-sHBZ or its mutants. After 24 hours, cells were treated with or without MG132 for 12 hours. Cell lysates were subjected to IP using anti-FLAG followed IB using anti-HA.