FIG. 1.

Interaction of NEMO with polyubiquitinated IRAK1 in IL-1-stimulated IL-1R cells. (A) IL-1R cells were transfected with a vector expressing either wild-type GST-NEMO or the GST-NEMO[D311N] mutant form. After 24 h, the cells were starved for 16 h, stimulated with 5 ng/ml IL-1β for the times indicated, and then extracted with lysis buffer. To precipitate proteins bound to GST-NEMO, an aliquot of cell extract (0.5 mg protein) was added to 20 μl of glutathione-Sepharose beads and after 1 h of incubation at 4°C, the beads were collected by centrifugation and washed three times with 1 ml of lysis buffer and once with 1 ml 10 mM Tris-HCl (pH 8). The bound proteins were released by denaturation in 1% SDS, subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotted with a variety of antibodies to detect the proteins indicated. In the third panel from the top, the band corresponding to unmodified TRAF6 is marked by an arrow and a band that cross-reacts nonspecifically with the anti-TRAF6 antibody and corresponds to the position of GST-NEMO is marked by an asterisk. In addition, the cell extracts (20 μg protein) were analyzed by SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with the antibodies indicated (bottom two panels). The small difference in IRAK1 expression in extracts from cells expressing GST-NEMO and GST-NEMO[D311N] was not observed in other experiments and is therefore not significant. (B) IL-1R cells were deprived of serum for 16 h, stimulated with 5 ng/ml IL-1β for the times indicated, and then extracted with lysis buffer. A 0.5-mg sample of cell extract was incubated for 45 min with an anti-IRAK1 antibody or control immunoglobulin G (IgG). Twenty microliters of protein G-Sepharose beads was added, and after another incubation of 45 min, the beads were collected by centrifugation and washed three times with 1 ml lysis buffer and once with 1 ml 10 mM Tris-HCl (pH 8). The bound proteins were released by denaturation in 1% SDS, subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotted with antibodies recognizing NEMO and IRAK1. To detect NEMO, horseradish peroxidase-coupled protein G was used instead of a secondary antibody because this protein comigrates with the IgG heavy chain; this explains why a faint signal is still detected at the positions of these proteins in the control IgG lane and at time zero. (C) IL-1R cells were deprived of serum for 16 h, stimulated with 5 ng/ml IL-1β for the times indicated, and then extracted with lysis buffer. IRAK1 (top three panels) was immunoprecipitated from 3 mg of cell extract protein as in panel B. The bound proteins were released by denaturation in 1% SDS, subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotted with antibodies recognizing IKKα, IKKβ, and IRAK1. In addition, the cell extracts (20 μg protein) were analyzed by SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies recognizing phosphorylated IKKα/IKKβ and GAPDH as a loading control (bottom two panels). Abbreviations: IP, immunoprecipitation; p-IRAK1, phosphorylated IRAK1.