Physical interaction between Tax and CYLD. (A) Cell lysates were prepared from the HTLV1-transformed C8166 cell line and subjected to IP using either a control pre-immune serum or anti-CYLD. The precipitated CYLD and its associated Tax were analyzed by IB. (B) HEK293 cells were transfected with HA-CYLD and Tax either in the absence (-) or presence (+) of IKKγ. The cell lysates were subjected to IP using anti-CYLD followed by IB to detect the precipitated CYLD and its associated Tax (top two panels). The cell lysates were also subjected to direct IB to monitor the expression of IKKγ and Tax. (C) HEK293 cells were transfected with HA-CYLD and Tax. The cells were stained with anti-HA (Y-11) and a mouse monoclonal anti-Tax antibody, followed with Texas red-conjugated donkey anti-rabbit Ig and FITC-conjugated donkey anti-mouse Ig. Cells were also counterstained with Hoechst 33258 for nuclear visualization. The expression of CYLD, Tax, Tax-CYLD merge, and nucleus (Hoechst) are shown. Note that the cytoplasmic, but not nuclear, Tax was colocalized with CYLD (Tax-CYLD merge, yellow color).