Gene by PF or 228 FI14 or DMSO as a contr The vehicle. After 24 hours the cells with 4% paraformaldehyde A-966492 in PBS were fixed. Next, the cells were washed with PBS and permeabilized with 0.2% Triton X-100 and 1% BSA in PBS. The cells were washed with PBS and then incubated with tetramethylrhodamine B isothiocyanate-labeled phallocentrism Dine. The cells were washed three times with PBS followed by incubation with 1 mg / ml Hoechst 33258 bisbenzimide in 1% BSA in PBS. The Objekttr hunters were mounted on Objekttr Happy with fluorescent mounting medium. Images were acquired with a 63 objective with a Zeiss Observer Z1 microscope and the AxioVision software. 2.8. Endothelial cells germination dishes tissue culture tests were renatured collagen I-coated to fibril Re collagen gels as above before incubation with radiolabelled ATP described form in the presence or absence of exogenous recombinant GST paxillin as a target substrate.
Kinase A-674563 Akt inhibitor reactions were incubated and proteins Subsequently End transmitted by SDS-PAGE and transferred to membranes. The membranes were exposed to develop embedded on a slide for the autoradiography signal of P32 in the phosphorylation reactions and were then subsequently End in Western blot analysis for FAK and subjected to a total recombinant paxillin, to ensure equal loading. FAK autophosphorylation was significantly by the presence of one or PF FI14 228 relative to the DMSO independently Ngig by the addition of exogenous paxillin to the kinase reaction.
In addition was the kinase activity of t against target substrates FAK, in this case exogenous recombinant paxillin, was also by the Pr Presence an FI14 or PF reduces the 228th Equivalent levels of exogenous FAK and paxillin included in the kinase reactions were also best by immunoblot analysis for each specific protein CONFIRMS. So it seems that small molecules as inhibitors are able to effectively inhibit FAK endothelial cells from FAK autophosphorylation and phosphorylation of kinases at concentrations of less than previously reported for other cell types. 3.3. The FAK inhibitor PF-573 228 induces apoptosis of endothelial cells Since our first study, beautiful PROTECTED lebensf of the number HIGEN cells, reduces the Lebensf Ability of the cells, we observed k nnten Due to decreased proliferation or increased Ht apoptosis.
We ma S apoptotic cells and the proportion of cells in various stages of the cell cycle found by flow cytometry of propidium iodide Rbten cells. HUVEC were treated with each inhibitor at various concentrations FAK in the presence of 50 ng / ml VEGF for 48 h, in which the cells were fixed permeabilized, and stained with propidium iodide Customised Rbt for FACS analysis incubated. We observed that exposure to PF 228 in consequence of increased Hten number of apoptotic HUVEC cells in a dose-ngigen As measured by the proportion of cells in the phase subG1 cell cycle, compared with contr the vehicle. Interestingly, no increase in apoptosis after treatment with FI14 at Hnlichen concentrations was observed. As to the proportion of cells in the G1 phase of the cell cycle, there was a tendency to reduce the amount of G1 in cells treated with 5 mM PF 228, which was simultaneously with the increase of apoptotic cells to decrease. However was no significant Ver Change in the percentage of cells in G1