The HCMEC TEER was measured using the Elec trical Resistance System, following the manufacturers instructions. KPT-185 In brief, coated inserts without cells were used as a blank. The electrical resistance of each in sert following treatment with TWEAK, P1. 17, or TNF was calculated by subtracting the blank from each reading. Inhibitors,Modulators,Libraries Each condition was run in duplicate, and the resistance measured twice for each well. Western blot analysis The following antibodies were used goat anti TWEAK, rabbit anti ZO 1, and mouse anti B actin. Protein concentrations were determined using the Lowry method. After boiling, aliquots containing equal amounts of protein were loaded in Laemmli buffer and separated by 8. 5% sodium dodecyl sulphate polyacrylamide gel electrophoresis Inhibitors,Modulators,Libraries using a MiniBlot system.

Proteins were transferred onto nitrocellulose membranes in transfer buffer. Membranes were incubated overnight in blocking buffer at 4 C and then probed with the primary antibody against ZO 1, B actin, or TWEAK diluted in blocking buffer. After washing, mem branes were incubated with a peroxidase conjugated sec ondary antibody. Finally, proteins were Inhibitors,Modulators,Libraries detected using a chemilumines cence kit. Films were digitized using GeneTools software, and optical densities of the bands were assessed using Scion Image software. In situ zymography To assess gelatinolytic activity of MMPs, hCMECD3 cells were grown on glass coverslips and the medium was supplemented to a final concentration of 5 mM CaCl2 and 10 ugml of intramolecularly Inhibitors,Modulators,Libraries quenched FITC labeled DQTM gelatin, as previously described.

After 2 h at 37 C in a humidified atmosphere containing 5% CO2, cells were rinsed in PBS, fixed with 4% paraformaldehyde for 5 min, and incubated for 5 min with DNA intercalant Hoechst 33258. Cells were observed with a Nikon Inhibitors,Modulators,Libraries E800 upright epifluorescence microscope and digital images were acquired at 1,024 1,024 pixels and saved in TIFF format. Fluorescence levels were measured at the level of individual cells using image J software image editing was performed using Adobe Photoshop. Gel zymography Standard methodology for gelatin zymography was used to detect MMP 2 and MMP 9 expression levels via their activity in cell supernatant or cell lysate samples, as described previously. Serum free culture superna tants and lysates were collected and protein concentra tion was normalized as mentioned above. Equal amounts of protein were subjected to 8% SDS PAGE containing 1 mgml gelatin in nondena turing, nonreducing conditions. After electrophoresis, gels were washed twice for 30 min in 2. 5% Triton X 100 to remove SDS and incubated for 48h in MMP activating buffer, 50 mM Tris HCl, pH kinase inhibitor Wortmannin 7. 5, with 10 mM CaCl2 at 37 C. Gels were then stained with 0.