We for that reason utilized flow cytometry to assess the total basal F actin content in the unique transfectants. Fig. 1C and 1D show the imply fluorescence of GFP transfectants, which didn’t substantially differ based on Students t test. As a manage, transfected cell had been pretreated with Cytochalasin D, a drug known to inhibit actin polymerization. Moreover, this experiment permitted us to calculate the transfection efficiency, which was esti mated as 60 70%, based on the evaluation with 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.
Additionally, N WASP deficient cells don’t kind pedestals. These observations prompted us to examine the phosphorylation status of cortactin in WT mouse embryonic fibroblasts, MEFs deficient selleck in N WASP and MEFs deficient in N WASP in which the pro tein was later restored by means of retroviral transduction. Initial, we performed Western blotting handle experiments to assess the expression of N WASP, cortactin and actin. Fig. 2A shows that EPEC induces phosphorylation of tyro sine 466 at 3 hours of infection in WT MEFs, as detected utilizing an antibody against phospho Y466 cortactin. This outcome was corroborated making use of a second phospho precise antibody. Unexpectedly, phos phorylation of tyrosine residue 466 was not induced in N WASP deficient cells.
This outcome suggests that tyrosine phosphorylation of cortactin for the duration of EPEC infection depends on the presence of N WASP. To confirm this, we infected R cells with EPEC and examined levels of read what he said phosphoY466 cortactin. Fig. 2A shows that N WASP re expression partially restored cortactin tyrosine phosphor ylation levels. In 3 independent experiments the nor malized typical induction was 1 0. 2 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. Offered the absence of cortactin tyrosine phosphorylation in EPEC infected N WASP deficient cells, we then checked Src activation, utilizing a commercially available phospho active Src antibody. Fig. 2B demonstrated that equal activation of Src was achieved for the duration of EPEC infec tion in all cell forms studied, although, as expected, the levels of total Src remained constant for the duration of infection. This outcome showed that the lack of cortactin phosphorylation in N WASP deficient cells was not because of a block in Src activa tion.