FIG 4.

ITC experiments and the effects of IKKγ-mimetic peptides on NF-κB activation. (A) (Top) ITC data obtained for the following: IKKγ titrated into GB1–ks-vFLIP in which both proteins were dialyzed into deionized water (left), an analogous experiment but with the proteins dialyzed into buffer (see Materials and Methods) (middle), and a competition assay in which spIKKγ was incubated with GB1–ks-vFLIP prior to titration of IKKγ (performed in deionized water) (right). (Bottom) Table showing the corresponding “apparent” K_ds and K_i for spIKKγ. (B) Immunoprecipitation assays involving Jurkat cell lysates in which ks-vFLIP was expressed in the presence and absence of GST-IKKγ using anti-IKKγ antiserum (upper image) and normal rabbit serum (lower image). GST-IKKγ prevents the association of ks-vFLIP with IKKγ, evidenced by the absence of ks-vFLIP from the pellet fraction. (C) (Top) Immunoprecipitation trials using Jurkat cell lysates treated with 50 μM spIKKγ in which ks-vFLIP was absent or overexpressed. (Bottom) Results of a kinase assay measuring the levels of phosphorylated IκB in the presence and absence of spIKKγ. (D) Similar to panel C except that immunoprecipitation assays were performed using BC3 cell lysates. The rabbit antiserum control is shown in the leftmost image.