Physiologic level of HBZ overcame the Tax-mediated suppression of TGF-β signaling. (A) Comparing the level of HBZ protein in HBZ-transfected HepG2 cells with the level of HBZ in ATL and HTLV-1–immortalized cell lines. Total protein was extracted from sHBZ-transfected HepG2 cells (samples from supplemental Figure 1) and the indicated cell lines, and subjected to immunoblotting using HBZ antibody. (B) Endogenous HBZ interacted with Smad3. ATL-55T cells were treated with 10 ng/mL TGF-β. After 10 hours, whole cell lysate was subjected to immunoprecipitation with anti-Smad3 or control IgG, and immunoprecipitates were probed with anti-HBZ antibody. (C) HBZ overcame the repression of TGF-β signaling induced by Tax. In 12-well plates, HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pCG-Tax (0, 0.2 μg), and pcDNA3.1-mycHis-sHBZ (0, 0.2 μg). At 24 hours after transfection, the cells were treated with or without 10 ng/mL TGF-β. After 24 hours, the cells were harvested and analyzed for luciferase activity. sHBZ and Tax were detected by Western blot (middle panel). CBB staining was shown as the loading control (bottom panel).