FIG. 4.

IRAK1 degradation is independent of the proteasome. (A) IL-1R cells were deprived of serum for 16 h, treated for 1 h with 25 μM MG-132 (Calbiochem) or DMSO as a control, stimulated with 5 ng/ml IL-1β for the times indicated, and then extracted with lysis buffer. An aliquot of the cell extract (20 μg of protein) was subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted (IB) with antibodies recognizing IRAK1, IκBα, Tpl2, ubiquitin, and GAPDH. (B) IRAK1 was immunoprecipitated (IP) from 0.5 mg of the cell extract protein used in panel A as described in the legend to Fig. 1. Beads were washed once with lysis buffer, twice with lysis buffer plus 500 mM NaCl, and once with 10 mM Tris-HCl (pH 8). Bound proteins were released from the beads by denaturation in 1% SDS, subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotted with antibodies recognizing ubiquitin and IRAK1. (C) IL-1R cells were deprived of serum for 16 h, stimulated with 5 ng/ml IL-1β for the times indicated, and extracted with lysis buffer containing either 1% Triton X-100 or 2% SDS. An aliquot of the cell extract (20 μg of protein) was subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies recognizing IRAK1 and GAPDH.