IRAK1 protein content was reduced in tolerant hTBE cells and remaining IRAK1 was not autophosphorylated after Ps. a. or IL-1β treatment. A) Proteins were harvested from day-21 hTBE cell cultures following apical challenge with Ps. a. filtrate at the times indicated. Equal amounts of protein were run per lane and subjected to western blot for IRAK1. B) Protein samples were obtained from cultures representing 3 different donors and IRAK protein content was examined as described in A, above. C and D) The apical surface of well-differentiated, day-21 hTBE cell cultures was challenged with Ps. a. filtrate, TSB or IL-1β as indicated and cultures were incubated for 24 hours. Following washing and basolateral media change, the apical surfaces were re-challenged as indicated and cellular protein was harvested 20 minutes following the second challenge. Equal amounts of protein per lane were immunoprecipitated with anti-IRAK1. Precipitates were subjected to in vitro kinase assay, run on polyacrylamide gels and exposed to a Phosphoimager screen. The results in panels C and D are representative of 3 separate experiments using cells derived from 3 different donors.