At 120 minutes fol lowing fluorescein addition, basal media was placed in a Corning 96 well black assay plate and fluores cein was determined using a Typhoon Trio Plus. Western blot analysis Western blot analyses are processed inhibitor licensed using the following protocol. Briefly, to prepare total cellular protein MDCK cells are washed with cold PBS and lysed in buffer con taining 1% Triton X 100, 1% sodium deoxycholate, 0. 1% SDS, 2 mM EDTA, 0. 15 M NaCl, 0. 01 M NaPO4, mini Complete protease inhibitor with the following phosphatase inhibitors 2 mM Na3VO4 and 10 mM NaF. DNA is sheared using a small gauge nee dle, and insoluble material is precipitated by centrifuga tion. Supernatants were collected and stored at 20 C for analyses. The method to prepare Triton X 100 soluble and insolu ble fractions was adapted from Singh et.

al. with minor modifications. MDCK cells were scraped into lysis buffer and incubated for 20 min. at 4 C. Following centrifugation, supernatants were collected and considered the TX 100 soluble frac tions, the pellets were placed in lysis buffer containing 1% SDS and TX 100 insoluble proteins were released by three sonication pulses using a Branson Sonifier 450. Insoluble material was removed by centrifugation, supernatants were collected and stored at 20 C for analyses. Protein concentration was determined using the Pierce BCA microtiter plate protocol using albu min as a reference standard. Lysates are denatured at 95 C for 5 min in Lammeli sample buffer, electrophoresed on 10% SDS PAGE gels, and electroblotted to PVDF mem brane for immunodetection.

Tight junction specific antis era including anti occludin and claudin 1 and 3 were employed in this study. Immunoblots are processed by blocking non specific binding sites in 5% non fat milk in Tris buffered saline with 0. 1% Tween 20 for 30 minutes followed by incubation with diluted primary antibody for 2 hours at room temperature. Immunoblots are then washed three times in TBS T fol lowed by incubation with an HRP conjugated secondary antibody. Following extensive washing with TBS T, immunoblots are developed with a stable West Pico chemiluminescent substrate. The image was captured on the VersaDoc 3000 and ana lyzed with the integrated QuantityOne 1 D analysis soft ware. Immunofluorescent analysis MDCK cell monolayers were grown on culture treated cover slips and treated for 24 hours in one of the follow ing conditions media only, TNF IFN, or TNF IFN with U0126. Layers were rinsed once Carfilzomib with sterile PBS and placed on ice for ten minutes. Cells were permeabilized with an actin stabilizing permeabilization buffer containing 0. 2% Tri ton X100, 100 mM KCl, 3 mM MgCl2, 1. 3 mM CaCl2, 25 mM sucrose, and 2 mM HEPES, pH 7. 1 for 2 min on ice.