RNF4-mediated ubiquitylation of Tax in vivo. (A) Western blot analysis of endogenous RNF4 expression in RNF4-specific shRNA and control shRNA stably expressing cell lines. The electrophoretic migration of RNF4 is indicated. (B) Analysis of purified S-TaxGFP from HA-ubiquitin–expressing cells. S-TaxGFP was affinity precipitated from transiently transfected control shRNA and RNF4-specific shRNA (RNF4 knockdown) cells. Also shown are samples from Tax-expressing RNF4 knockdown cells that have been cotransfected with RNF4-mCherry or with RNF4-mCherry RM. (C) The same blot was probed with anti-HA Ab. (D) In vitro ubiquitylation of Tax by RNF4. Ubiquitylation reactions were assembled using recombinant, purified MBP-RNF4, SUMO-Tax, and Ubc4. The ubiquitylation assays were halted and separated by SDS PAGE and probed with anti-Tax Ab. Control reactions were assembled in the absence of ATP (−ATP), Ubc4 (−E2), MBP-RNF4 (−E3), or SUMO-Tax (−SUMO-Tax) as indicated. An ubiquitylation reaction with all components (Complete rxn) was also conducted. High-molecular weight adducts corresponding to ubiquitylated Tax is indicated (Ub_n-Tax). Unmodified Tax protein (Tax) and molecular weights (in kDa) are indicated.