Fig. 7.

Blocking of antibody epitopes by ubiquitin chains. (A) The experiment was performed as in Fig. 5B, except that immunoblotting was carried out using two different anti-IRAK1 antibodies (polyclonal H-273 and monoclonal F-4). (B) As in A, but after stimulation with IL-1β for the times indicated, cell extracts (20 μg protein) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies shown. The antibody against GAPDH was used as a loading control. (C) As in B, except that IRAK1 was immunoprecipitated from the cell extracts (1 mg protein) and incubated without (control) or with USP2 plus λ-PPase. Samples were subjected to SDS-PAGE and IRAK1 was visualised by immunoblotting for 5 s (“short”) (upper panel) or 60 s (“long”) (lower panel) with an anti-IRAK1 antibody.