We hence utilised flow cytometry to assess the total basal F actin content material of your different transfectants. Fig. 1C and 1D show the mean fluorescence of GFP transfectants, which didn’t substantially differ based on Students t test. As a handle, transfected cell were pretreated with Cytochalasin D, a drug known to inhibit actin polymerization. Moreover, this experiment allowed us to calculate the transfection efficiency, which was esti mated as 60 70%, depending on the analysis on the expression of GFP constructs. EPEC induces N WASP dependent tyrosine phosphorylation of cortactin Contrary to the transient phosphorylation induced by EHEC, EPEC infection of CH7 mouse fibroblasts induces tyrosine phosphorylation of cortactin. Src has been shown to phosphorylate cortactin on tyrosines Y421, 466 and 482, which decreases cortactin affinity for N WASP in vitro.
Furthermore, N WASP deficient cells usually do not type pedestals. These observations prompted us to examine the phosphorylation status of cortactin in WT mouse embryonic fibroblasts, MEFs deficient get more information in N WASP and MEFs deficient in N WASP in which the pro tein was later restored by way of retroviral transduction. Initially, we performed Western blotting manage experiments to assess the expression of N WASP, cortactin and actin. Fig. 2A shows that EPEC induces phosphorylation of tyro sine 466 at three hours of infection in WT MEFs, as detected utilizing an antibody against phospho Y466 cortactin. This result was corroborated using a second phospho particular antibody. Unexpectedly, phos phorylation of tyrosine residue 466 was not induced in N WASP deficient cells.
This outcome suggests that tyrosine phosphorylation of cortactin through EPEC infection depends upon the presence of N WASP. To verify this, we infected R cells with EPEC and examined levels of selleck Omecamtiv mecarbil phosphoY466 cortactin. Fig. 2A shows that N WASP re expression partially restored cortactin tyrosine phosphor ylation levels. In three independent experiments the nor malized typical induction was 1 0. two for WT cells, 0 for N WASP deficient cells and 0. 5 0. 1 for R cells. This sup ports the idea that EPEC induced tyrosine phosphoryla tion of cortactin in cells calls for N WASP. Given the absence of cortactin tyrosine phosphorylation in EPEC infected N WASP deficient cells, we then checked Src activation, utilizing a commercially obtainable phospho active Src antibody. Fig. 2B demonstrated that equal activation of Src was achieved through EPEC infec tion in all cell types studied, while, as anticipated, the levels of total Src remained constant in the course of infection. This outcome showed that the lack of cortactin phosphorylation in N WASP deficient cells was not resulting from a block in Src activa tion.

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