IκBα degradation, MAP kinase activation and induction of nuclear NF-κB or AP-1 transcription factors were strongly attenuated following a second Ps. a. filtrate challenge of tolerant hTBE cells. The apical surface of well-differentiated, day-21 hTBE cell cultures was challenged with Ps. a. filtrate or TSB as indicated and cultures were incubated for 24 hours. Following washing and basolateral media change, the apical surfaces were re-challenged as indicated and proteins were harvested for IκBα and βactin (A) or phospho- and total MAP kinase (B) western blot analysis 20 minutes later. 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. C) hTBE cells were re-challenged as indicated and nuclear proteins were harvested 2 hours following the second challenge. Equal amounts of nuclear protein per lane were subjected to EMSA using oligonucleotide probes for NF-κB or AP-1, only the shifted bands are shown. Similar results were obtained in 3 separate experiments with cell cultures from 3 different donors.