Figure 2

Ps. a. filtrate induced IRAK1 autophosphorylation, IκBα degradation and MAP kinase phosphorylation. A)Proteins were harvested from day-21 hTBE cell cultures treated apically with 20% Ps. a. filtrate or from THP1 monocytic cells treated with LPS at the times indicated. Equal amounts of protein were immunoprecipitated with anti-IRAK1 or control IgG for each cell type. Precipitates were subjected to in vitro kinase assay and polyacrylamide gel electrophoresis and exposed to a Phosphoimager screen. B and C) Day-21 hTBE cell cultures were treated with Ps. a. filtrate, and harvested for western blot of IκBα (B) or phospho- and total MAP kinases (C) at the indicated time points. Equal amounts of protein were run per lane on each gel. The βactin and total MAP kinase western blots represent re-probing of the IκBα and phospho-MAP kinase blots, respectively. The results are representative of 3 separate experiments with cells derived from 3 different donors.