A DNA vector encoding HA-Pellino 2 was transfected into IRAK1-null IL-1R cells with either wild-type IRAK1 (lane 1) to activate Pellino 2 (shown by a decrease in mobility using an anti-HA antibody) or with catalytically-inactive–IRAK1[K239A] (lane 2) as a control. 24 hours later the cells were lysed and 25 μg of extract protein was subjected to SDS/PAGE, transferred to nitrocellulose membrane and immunoblotted with the phospho-specific antibody that recognises phosphorylated Thr288 of Pellino 1 and the equivalent residue in Pellino 2 (Thr290). Binding of the primary antibody was detected using rabbit peroxidase conjugated anti-sheep IgG antibody (1 in 10, 000 dilution, Pierce). NS, nonspecific band recognised by the p-Pellino antibody. The cell extract was also immunoblotted with an anti-tubulin antibody as a loading control.