1% Triton X 100. The cells were incubated with a 1% solution of BSA, and stained with Rhodamine phalloidin. Stained F actin was visua lized using an OLYMPUS XB 51 fluorescence inverted microscope under 200 fold magnification. Immunoblot analysis Protein samples were sub jected to 8% or 12% SDS PAGE, and Nutlin-3a the proteins were then electrophoretically transferred to a polyvinylidene fluoride membrane blocked by 5% BSA for 1 h at room temperature and then incubated with antibodies overnight at 4 C. Secondary antibody was incubated for 1 h at room temperature. A chemiluminescence reagent, ECL western blotting detection reagent, was used to make the labeled protein bands visible. The blots were developed by the enhanced chemilumines cence method.

Phosphorylation of p115RhoGEF After serum deprivation for 6 h, BMECs were labeled with 150 uCiml 32P for 4 h in phosphate free MEM. Cells were then stimulated with TNF a for Inhibitors,Modulators,Libraries the indi cated times, quickly transferred onto ice, washed with ice Inhibitors,Modulators,Libraries cold PBS containing 500 uM Na3VO4 and lysed. After centrifugation, the cleared lysate Inhibitors,Modulators,Libraries was incubated with either control IgG or anti mouse P115RhoGEF Ab for 2 h fol lowed by the addition of protein AG plus agarose beads overnight. The beads were then collected by centrifuga tion, washed with detergent free buffer and 2 ugmL each of pepstatin A, leupeptin, and aprotinin. The above proce dures were performed at 4 C. Protein from each sample was eluted by boiling the beads in SDS sample buffer, elec trophoresing on 8% SDS polyacrylamide gels, and transfer to nitrocellulose for visualization of p115RhoGEF phos phorylation by autoradiography, followed by western blot ting with p115RhoGEF antibody to verify equal protein loading.

Specificity of the p115RhoGEF antibody was con firmed using normal mouse IgG as a negative Inhibitors,Modulators,Libraries control. Statistical analyses All of the data are expressed as the meansSD. A Stu dents t test was performed to determine the significant difference between two groups. One way ANOVA ana lysis followed by Inhibitors,Modulators,Libraries Student Neuman Keuls post hoc tests was utilized to determine the significant differences among multiple groups. P 0. 05 was considered to be statistically significant. Results TNF a activates RhoA, mediating barrier dysfunction in Bend. 3 cells To address the direct involvement of RhoA in TNF a induced Bend. 3 cell barrier permeability, n19RhoA cells were used to sup press activation of RhoA.

The remarkable sellckchem inhibitory effect of n19RhoA was confirmed by pull down assay. TNF a exposure induced rapid and pro longed RhoA activation in a time course manner. RhoA activity increased significantly at 1 min, with a maxi mum response occurring at 30 min followed by a decline at 3 h. However, RhoA activity remained higher than the baseline even 12 h after TNF a administration.