Tax undergoes K63 ubiquitination, which is inhibited by CYLD. (A) 293 cells were transfected with HA-tagged ubiquitin together with vector control (V) or Tax. Protein lysates were subjected to IP using anti-Tax, and ubiquitin-conjugated Tax and Tax were detected by IB with HRP-anti-HA and anti-Tax, respectively. (B) 293 cells were transfected with Tax together with the indicated ubiquitin mutants. Tax was subject to IP and ubiquitination assays as in A (left panel). Cell lysates were analyzed by IB to monitor ubiquitin expression (right panels). K48R and K63R harbor K/R substitutions at lysines 48 and 63, respectively. K48 and K63 harbor K/R substitutions in all lysines except 48 and 63, respectively. (C) HEK293 cells were transfected with Tax and HA-tagged ubiquitin either the in the absence (-) or the presence of wildtype (WT) CYLD or a catalytically inactive CYLD mutant (Mut). Cell lysates were subjected to Tax ubiquitination (left panel) and IB (right panel) assays. The Tax expression level was comparable between the different lanes (data not shown).