The p110𝛼 and p110𝛽 Isoforms of Class I Phosphatidylinositol 3-Kinase Are Involved in Toll-Like Receptor 5 Signaling in Epithelial Cells

Figure 1

Activation of PI3K in Caco-2 cells by E. coli flagellin. (a) In vitro kinase assay. Caco-2 cells treated with flagellin (FliC) 1 $\mu$g/ml for the indicated times were lysed and immunoprecipitated with anti-p85. An aliquot of each immunoprecipitate was analyzed by Western blot for p85, and the remainder was reacted with phosphatidylinositols and $\gamma$-${}^{32}\text{P}$-ATP and separated by thin-layer chromatography. The PIP3 band is indicated by the arrow. Densitometry performed on autoradiograms from three independent experiments is shown below. ${}^{*}P<.05$, FliC versus control (t-test). (b) Tyrosine phosphorylation of PI3K. Caco-2 cells treated with 500 ng/ml flagellin for the indicated times were lysed and immunoprecipitated with anti-p85. Precipitates were separated by SDS-PAGE and Western blots performed using 4G10 antiphosphotyrosine. Blots were stripped and reprobed with anti-p85 to confirm equal efficiency of immunoprecipitation. Results are representative of two separate experiments.

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