2% SDS, 150 mM NaCl, 2 mM EDTA, 10 mM HEPES, pH 7.3) that contained 20 mM NaF, 1 mM orthovanadate and a protease inhibitor cocktail (1100 dilution) to www.selleckchem.com/products/Tubacin.html obtain total cell lysates. The homogenized lysates were centrifuged at 10,000��g for 10 min at 4��C. The supernatants were collected and stored at ?20��C for later analysis. Equal amounts of protein (30 ��g/lane) per sample were separated by SDS-PAGE electrophoresis on 13% gels and transferred to nitrocellulose sheets. The membranes were blocked and incubated with primary antibodies against claudin-1 (1500), claudin-3 (2 ��g/mL), or ��-tubulin (11000). After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies. The proteins were visualized using an enhanced chemiluminescence kit (GE Healthcare, Chalfont St.

Giles, UK). The bands were quantified according to their optical densities using LabWorks 4.6 (Bio-Rad Laboratories, Hercules, CA). Immunofluorescence Caco-2 (2��105) and HT-29 (105) cells were seeded on glass coverslips in 24-well plates and incubated until confluence. The cell monolayers were subsequently washed with PBS and fixed with methanol for 10 min at ?20��C. Next, the cells were blocked with 0.2% BSA in PBS for 1 h and permeabilized with 0.1% Triton X-100. The cells were incubated overnight with anti-claudin-1, anti-claudin-3 (140), anti-E-cadherin (1300) antibodies, followed by 1 h incubations with the appropriate Alexa 488-conjugated secondary antibodies (1500). Following the incubations, the cells were mounted using n-propyl-gallate to allow for visualization with either an Axio Observe.

Z1 microscope that was equipped with an AxioCam HRc and the AxioVision Release 4.8 digital image processing software (Carl Zeiss Inc., Jena, Germany) or a confocal laser scanning microscope FV10i-O, from which images were analyzed using the FV10-ASW software (Olympus, Tokyo, Japan). Wound Healing Assay HT-29 cells (2��105) were seeded into 6-well plates and incubated until confluence. To perform wound-healing assays, cell monolayers were manually wounded by scraping with pipette tips. After different treatments, the cells were permitted to migrate into the denuded areas and photographed immediately after wounding (0 h) and at 6 h or 24 h after wounding. The distance between the two edges of a denuded area was quantified in triplicate and repeated independently three times.

Migration is represented as the percentage of cell migration and plotted on a graph. Cell Proliferation Assays The relative viable cell numbers were determined using crystal violet or trypan blue dyes. The crystal violet method was conducted as described previously [31]. Briefly, the HT-29 cells (104) were seeded into 96-well plates and incubated in culture medium with or without EGF for 24 and 48 h before ethanol fixation for 10 min. A crystal violet solution (0.05% crystal violet and 20% methanol) was added to the cells for Brefeldin_A 10 min.