Direct interaction between Tax and Sp1. A, Tax does not alter Sp1 level. HeLa/18 × 21-EGFP cells were transfected with a control plasmid, pcDNA3 (Ctrl), or pBC12-Tax (Tax), using the calcium phosphate method. Immunoblotting was used to detect Sp1, Tax, p21, and β-Actin in the whole cell lysates at 48 h post-transfection. B, Tax associates with Sp1 in transfected HEK293 cells. HEK293 cells were transfected with pcDNA3 (-) or pBC12-Tax (+) as in (A). Cell lysates were immunoprecipitated with Sp1 or Tax antibody and immunoblotted with the same. C, Tax associates with Sp1 in HTLV transformed MT4 T cells. Jurkat and MT4 cell lysates were immunoprecipitated with Sp1 or Tax antibody and immunoblotted with the same as indicated. D, Tax binds Sp1 in vitro. Approximately 200 ng of Sp1 (Promega), and 30 μl of Ni-NTA agarose beads were mixed with (+) or without (-) purified hexa-histidine-tagged Tax (TaxH6) (~2 μg) as described in METHODS. The beads were collected by centrifugation and washed with the binding buffer. The bound proteins were eluted in SDS sample buffer and analyzed by immunoblotting (Sp1) and Coomassie blue staining (Tax).