FIG. 1.

Tetrameric oligomerization of IKKγ. (A) IKKγ-deficient 1.3E2 cells and 1.3E2 cells stably expressing IKKγ were metabolically labeled with [³⁵S]methionine. Lysates were treated with EGS, IKKγ was immunoprecipitated (IP: IKKγ), and pellets were washed and analyzed by SDS-PAGE and autoradiography. To confirm that the 200-kDa band resulting from the EGS cross-link consists exclusively of IKKγ, the band was excised from a polyacrylamide gel, and as indicated, proteins were electroeluted and treated with hydroxylamine-HCl to cleave EGS. IKKγ complexes were precipitated and separated by SDS-PAGE, and components were detected with a phosphorimager (top gels). The lack of immunoprecipitated labeled proteins in 1.3E2 cells confirms the specificity of the antibody. IKKγ expression before cross-linking was determined by Western blotting (WB). (B) Metabolically labeled HeLa cells were lysed, and proteins were cross-linked with sulfo-EGS. Immunoprecipitation with an IKKγ antibody (IP: IKKγ) was performed with (+) or without (−) preblocking with a specific peptide, as indicated above the gel. Products were analyzed by SDS-PAGE and autoradiography. The arrows in panels A and B indicate the position of IKKγ tetramers. (C) [³⁵S]methionine-labeled, N-terminally FLAG-tagged IKKγ (FLAG-IKKγ) and C-terminally HA-tagged IKKγ (IKKγ-HA) were prepared by coupled in vitro transcription and translation and cross-linked with EGS. The reaction was stopped by the addition of Tris, and immunoprecipitations (IP) with the indicated antibodies were performed. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gel. (D) (Left) Recombinant IKKγ was purified from E. coli and analyzed on a Coomassie blue-stained SDS-polyacrylamide gel along with protein molecular mass markers, as indicated. (Right) Analytical ultracentrifugation analysis of purified recombinant IKKγ (Superose 6 peak fractions). Radial concentration distribution curves of IKKγ dissolved in 20 mM Tris-HCl (pH 7.5) containing 100 mM NaCl are shown. The profiles were recorded at 10,000 rpm and wavelengths of 220 (○), 225 (•), or 230 (□) nm. The three curves were fitted globally using the polymole program, resulting in a molecular mass of 192.6 ± 3.6 kDa.