RNF4 binds Tax. HEK 293T cells were transiently transfected to express SGFP, S-TaxGFP, or S-TaxGFP mutants with RNF4-GFP. At 48 hours after transfection, S-tagged protein complexes were isolated on S-protein agarose, separated by SDS-PAGE, and subjected to immunoblot analysis with a anti-GFP polyclonal Ab. (A) Affinity precipitation of S-TaxGFP (lanes 1 and 2), SGFP (lanes 3 and 4), or S-TaxK280/284RGFP (lanes 5 and 6) in the presence or absence of cotransfected RNF4-GFP as indicated are shown. As a control for binding specificity, we also incubated S-beads with RNF4-GFP alone (lane 7). Shown is the immunoblot analysis with a polyclonal anti-GFP Ab. The migration of each GFP-fusion protein is indicated (arrows). Whole-cell lysates were normalized for expression of RNF4 (RNF4 input). (B) Affinity purification of S-TaxGFP and deletion mutant S-TaxΔ203-254GFP in the presence or absence of cotransfected RNF4-GFP. Indicated are the Tax/Tax mutant and RNF4 (arrows). (C) Relative binding efficiency of Tax and Tax mutants to RNF4. Densitometry analysis was used to determine the relative amount of RNF4 that coprecipitated with each of the Tax mutants. Binding efficiency was expressed as the ratio between recovered RNF4 and Tax or Tax mutant after subtraction of background. The binding of wild-type Tax to RNF4 was set at 100% and all other measures of binding efficiency were compared with this ratio.