Figure 4.

PD blocks tyrosine phosphorylation of EPEC Tir but not Tir localization. (a–d) Images of 3T3 cells treated with PD166326 (10 μM) and exposed to EPEC. Cells were stained with DAPI (a) to recognize EPEC, with FITC-phalloidin to recognize actin (b), with α-Tir pAb (c) and with α-phosphotyrosine mAb 4G10 (d). Note that Tir is present despite absence of pedestals and that phosphotyrosine staining is not evident beneath attached EPEC. Bar, 5 μm. (e–g) Tyrosine phosphorylation of EPEC Tir can be blocked or reversed by PD. Cells were treated with DMSO (e) or PD (f) and were left uninfected (0 h) or infected with EPEC for the times indicated. Cells were lysed and subjected to Western analysis with α-phosphotyrosine mAb 4G10 (top), stripped, and then reprobed with α-Tir pAb (bottom). Note that Tir protein is evident after 3 h and becomes phosphorylated in DMSO-treated cells and that PD blocks Tir phosphorylation. (g) Cells were left uninfected (lane 1) or infected with EPEC for 6 h, treated with PD for the times indicated, and analyzed as in e and f. Note the band corresponding to Tir becomes dephosphorylated within 5 min of adding PD. (h–o) Cells were infected with EPEC and stained with DAPI to recognize bacteria (h and l), with FITC-phalloidin to recognize actin (I and m), and with α-Tir pAb (j) or α-4G10 mAb (n) to recognize phosphotyrosine. In the merged images (k and o), bacteria are pseudocolored blue, Tir or phosphotyrosine red, and actin green. Bar, 5 μm.