HBZ interacts with Smad proteins. (A) HBZ interacted with Smad proteins. COS7 cells were cotransfected with mycHis-sHBZ (6 μg) and FLAG-Smad2, Smad3, Smad4, and Smad7 (6 μg). At 24 hours after transfection, the cells were treated with or without TGF-β (5 ng/mL). Cell lysates were subjected to immunoprecipitation using anti-c-Myc followed by immunoblotting using anti-FLAG for detection of Smad proteins. (B) sHBZ colocalized with Smad3 in the cell nucleus. COS7 cells were transfected with mycHis-sHBZ (0.6 μg) together with (v-viii) or without (iii-iv) FLAG-Smad3 (0.4 μg). sHBZ was detected using anti-MYC Cy3 antibody (iv,vi). Smad3 was detected using anti–Flag-biotin and secondary streptavidin-Alexa-488 antibody (ii,v). The overlay of sHBZ and Smad3 is shown (vii-viii). DAPI (4,6-diamidino-2-phenylindole) was used to counterstain the nucleus (i,iii). (C) HBZ did not influence the Smad3/Smad4 interaction. COS7 cells were transfected with the indicated expression vectors (3 μg each). Cell lysates were subjected to immunoprecipitation using anti-FLAG followed by immunoblot (IB) using anti–c-Myc.