Fig. 6. Induction of p100–IKKα binding by Tax. (A) Stable association of p100 with IKKα in HTLV-infected T cells. Cell extracts were prepared from mitogen-stimulated Jurkat cells or the HTLV-infected C8166 cells and subjected to IP using either a preimmune serum (PI) or anti-p100 antibody, and the coprecipitated IKKα was detected by IB (lanes 1–4). The extracts were also analyzed directly by IB to detect the expression levels of IKKα, p100, Tax and actin (lanes 5 and 6). (B) IKKα, but not IKKβ, is in the p100 complex in HTLV-infected T cells. The C8166 cell lysates were subjected to IP using the indicated PI and immune sera. The p100 (upper panel) and Tax (lower panel) proteins in the immune complexes were analyzed by IB. (C) Binding of transfected p100 to endogenous IKKα in Tax-expressing cells. Parental Jurkat or its IKKγ-deficient variant was transfected with the pcDNA vector (V) or the expression vectors indicated below the figure (1 µg for Tax, 0.2 µg for HA-tagged IKKγ and 1 µg for p100). The p100–IKKα (top panel) and p100–Tax (second panel) interactions were analyzed by coIP. The lower three panels show IB analyses of the expression levels of p100, IKKα and IKKγ in the cell lysates. The larger molecular size of the exogenously transfected IKKγ (bottom panel, lanes 5 and 6) was due to its HA epitope. (D) Induction of p100 association with IKKα but not IKKβ by Tax. 293 cells were transfected with the expression vectors indicated (0.25 µg for IKKα and IKKβ, 1 µg for Tax, 0.5 µg for p100). All the cells also received the IKKγ expression vector (0.2 µg), since endogenous IKKγ is low in these non-lymphoid cells (Xiao et al., 2000). The p100–IKKα and p100–IKKβ interactions were analyzed by coIP (top panel), and the protein expression was analyzed by IB (lower panels). (E) Kinase-dead IKKα and IKKβ do not support Tax-induced p100 processing. The transfection was performed as in (D), followed by IB analysis of p100/p52 (upper), HA-tagged IKK mutants and Tax.