Tax induces nuclear accumulation and SUMOylation of NEMO. (A) HeLa cells were transfected with empty vector, Tax, or Tax mutants K4-8R, K4-8R-Ub, or K4-8R-SUMO1 plasmids. Cell fractionation was performed as described in the Methods. Lysates from the nuclear fraction (N), the soluble cytosolic fraction (C), and the intermediate fraction (I), were blotted with different antibodies as indicated. (B) Cell fractionation was performed on HTLV-I–negative Jurkat cells and on HTLV-I–infected HuT-102 cells as described in panel A. (C) 293T and HeLa cells were cotransfected with His-NEMO and Tax plasmids and proteins purified by denaturing Ni-NTA pull-down were blotted with mAb against polyubiquitylated proteins (FK2) and a polyclonal antibody against NEMO (top panel). Lysates were blotted with mAb against Tax or GAPDH (bottom panel). (D-F) 293T or HeLa cells were cotransfected with His-NEMO, HA-SUMO1, and Tax plasmids or the Tax mutants K4-8R, K6-8R, K4-8R-Ub, K4-8R-SUMO1 plasmids, and proteins purified by denaturing Ni-NTA pull-down were blotted with mAb against HA and a polyclonal antibody against NEMO. Lysates were blotted with mAb against Tax or GAPDH.