Figure 6
Figure 6. WWOX selectively inhibits Tax-mediated IKKα recruitment into RelA and subsequent RelA S536 phosphorylation. (A) Interaction between Tax and WWOX. The 293 cells were transfected with Tax alone or together with Myc-WWOX, followed by IP using Myc antibody and IB using Tax antibody. The inputs of Myc-WWOX and Tax were analyzed by direct IB. (B) In vivo interaction between Tax and WWOX. Endogenous Tax proteins of the indicated HTLV-I–transformed T cells were pulled down by Tax antibody, and the endogenous WWOX proteins that bound to Tax were examined by WWOX antibody. Jurkat cells were used as a negative control. The inputs of endogenous WWOX and Tax were analyzed by direct IB. (C) Interaction between Tax and WWOX Y33R. 293 cells were transfected with Myc-WWOX wild-type or its Y33R mutant in the presence or absence of Tax, followed by IP using Tax antibody and IB using Myc antibody. The inputs of Tax, Myc-WWOX, and its Y33R mutant were analyzed by direct IB. (D) Effect of WWOX on Tax stability. The 293 cells were transfected with Tax in the presence or absence of Myc-WWOX or Myc-WWOX Y33R, and were mock treated or treated with CHX alone or CHX plus MG132 for 5 hours. The expression levels of Tax, Myc-WWOX, Myc-WWOX Y33R, and Hsp90 were detected by IB. (E) Effect of WWOX on Tax-induced p100 processing. The 293 cells were transfected with the indicated plasmids, followed by IB to examine protein levels of p100/p52, Tax, WWOX, and its Y33R mutant. (F) Effect of WWOX on Tax-induced IκBα phosphorylation and degradation. HeLa cells were transfected with Tax in the presence or absence of Myc-WWOX or Myc-WWOX Y33R, followed by IB to examine phosphorylation-modified or unmodified phosphorylated IκBα. Protein levels of Tax, Myc-WWOX, and its Y33R mutant were also examined. (G) Selective inhibition of Tax-mediated RelA S536 phosphorylation by WWOX. The 293 cells were transfected with Tax in the presence or absence of Myc-WWOX or Myc-WWOX Y33R followed by IB to examine S536 phosphorylation of endogenous RelA. (H) Selective inhibition of Tax-mediated IKKα recruitment to RelA by WWOX. The 293 cells were transfected with the indicated plasmids followed by IP using IKKα specific antibody (lanes 2-6) or control IgG (lane 1) and IB using HA antibody or IKKα antibody. The inputs of all related proteins were also shown.

WWOX selectively inhibits Tax-mediated IKKα recruitment into RelA and subsequent RelA S536 phosphorylation. (A) Interaction between Tax and WWOX. The 293 cells were transfected with Tax alone or together with Myc-WWOX, followed by IP using Myc antibody and IB using Tax antibody. The inputs of Myc-WWOX and Tax were analyzed by direct IB. (B) In vivo interaction between Tax and WWOX. Endogenous Tax proteins of the indicated HTLV-I–transformed T cells were pulled down by Tax antibody, and the endogenous WWOX proteins that bound to Tax were examined by WWOX antibody. Jurkat cells were used as a negative control. The inputs of endogenous WWOX and Tax were analyzed by direct IB. (C) Interaction between Tax and WWOX Y33R. 293 cells were transfected with Myc-WWOX wild-type or its Y33R mutant in the presence or absence of Tax, followed by IP using Tax antibody and IB using Myc antibody. The inputs of Tax, Myc-WWOX, and its Y33R mutant were analyzed by direct IB. (D) Effect of WWOX on Tax stability. The 293 cells were transfected with Tax in the presence or absence of Myc-WWOX or Myc-WWOX Y33R, and were mock treated or treated with CHX alone or CHX plus MG132 for 5 hours. The expression levels of Tax, Myc-WWOX, Myc-WWOX Y33R, and Hsp90 were detected by IB. (E) Effect of WWOX on Tax-induced p100 processing. The 293 cells were transfected with the indicated plasmids, followed by IB to examine protein levels of p100/p52, Tax, WWOX, and its Y33R mutant. (F) Effect of WWOX on Tax-induced IκBα phosphorylation and degradation. HeLa cells were transfected with Tax in the presence or absence of Myc-WWOX or Myc-WWOX Y33R, followed by IB to examine phosphorylation-modified or unmodified phosphorylated IκBα. Protein levels of Tax, Myc-WWOX, and its Y33R mutant were also examined. (G) Selective inhibition of Tax-mediated RelA S536 phosphorylation by WWOX. The 293 cells were transfected with Tax in the presence or absence of Myc-WWOX or Myc-WWOX Y33R followed by IB to examine S536 phosphorylation of endogenous RelA. (H) Selective inhibition of Tax-mediated IKKα recruitment to RelA by WWOX. The 293 cells were transfected with the indicated plasmids followed by IP using IKKα specific antibody (lanes 2-6) or control IgG (lane 1) and IB using HA antibody or IKKα antibody. The inputs of all related proteins were also shown.

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