EPEC infection induces tyrosine phosphorylation of Crk adaptor proteins.

<p>HeLa cells were serum-starved for 16 h prior to infection with preactivated EPEC for 1, 2 or 3 h at an MOI of 45. The basal level of phosphorylation of Crk proteins was visualized in uninfected cells (- EPEC). (<b>A</b>) CrkII or IgG isotype control immunoprecipitates were probed by WB with a phosphospecific Ab against phospho-Tyr221 in CrkII (phospho-CrkII) to show induction of phosphorylation. The blot was also probed with anti-CrkI/II MoAb to show total levels of CrkII. The ratio of phospho-CrkII to non-phosphorylated CrkII in a representative experiment is shown. (<b>B</b>) Statistical analysis of the ratio of phospho-CrkII to CrkII signals with respect to the basal level in uninfected cells using one-way ANOVA with Dunnett test. The graph shows mean ± SD for four independent experiments. a.u.: arbitrary units. ***, p<0.001. (<b>C</b>) WBs with a phosphospecific Ab against phospho-Tyr207 in CrkL to show the induction of phosphorylation, and with anti-CrkL Ab to show total levels of CrkL. The ratio of the phospho-CrkL to CrkL in a representative experiment is shown. (<b>D</b>) Statistical analysis of the ratio of phospho-CrkL to CrkL levels with respect to the basal level at 0 h using one-way ANOVA with Dunnett test. The graph shows mean ± SD for four independent experiments. a.u.: arbitrary units. *, p<0.05. (<b>E</b>) Immunofluorescence images of HeLa cells that were starved for 16 h prior to infection with preactivated EPEC for 3 h at an MOI of 15. Immunofluorescence staining was done using phosphospecific Abs against Tyr221 in CrkII (phospho-CrkII) or Tyr207 in CrkL (phospho-CrkL) followed by Alexa 488-conjugated goat anti-rabbit secondary Ab (green). Actin was stained red using TRITC-phalloidin; bacteria were stained blue using DAPI. Pictures were taken on a confocal microscope and images from one section are shown, together with 4× digital zoom images (insets). Images were merged using Leica software.