Tax is SUMO-conjugated in the nucleus. (A-D) 293T (A-C) or Jurkat (D) cells were transfected with the plasmids encoding for Tax-6His or Tax mutants in the presence of HA-SUMO1 (A,D), HA-SUMO2 (B,D), or HA-SUMO3 (C,D) plasmids. Proteins purified by denaturing Ni-NTA pull-down were blotted with anti-HA (top panel) or anti-Tax (bottom panel) mabs. (E) HeLa cells were cotransfected with Tax and either Ub-HA and/or HA-SUMO3 plasmids. After immunoprecipitation with anti-Tax mab, recovered proteins were blotted with anti-Tax or anti-HA antibodies. Cell fractionation was performed as described in “Materials and methods.” Total lysates (left panels), nuclear extracts (lamin positive, tubulin negative; middle panels), and cytoplasmic extracts (lamin negative, tubulin positive; right panels) are displayed. Lysates were blotted with anti-HA and antitubulin antibodies to ensure equal loading.