The membranes were washed 3 times with TBS-T and then immunoreactive bands were visualized using ECL Western Blotting detection reagents (GE

Healthcare, Uppsala, Sweden) or Immuno Star LD (Wako). The membranes were Selleck SCH772984 stripped and probed with anti-β-actin antibodies as a loading control. GST-R5BD pull-down assay The GST-R5BD pull-down assay buy ABT-263 was based on the method described by Liu et al. [60]. Ca9-22 cells were transfected with GFP-Rab5 (WT) using Lipofectamine 2000 reagent, as described by the manufacturer (Invitrogen). The transfectants were pretreated with MAP kinase inhibitors, including a p38 inhibitor (SB203580, 5 μM), JNK inhibitor (SP600125, 1 μM), and ERK inhibitor (PD98059, 5 μM) (Calbiochem, San Diego, CA), or with an NF-κB inhibitor (PDTC, 5 μM) (Sigma-Aldrich, St. Louis, MO) at 37°C for 1 h followed by stimulating with 10 ng/ml TNF-α for 3 h. Thereafter, cell extracts were prepared in lysis buffer containing 25 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% Nonidet P-40, 2% glycerol, 1 mM dithiothreitol, and protease inhibitors. The cell lysates were centrifuged at 13,000 × g for 10 min at 4°C, and then the

supernatants were incubated with 20 μl of GST-R5BD bound to glutathione-Sepharose 4B beads for 10 min at 4°C under rotation. Thereafter, beads were collected and washed 3 times with lysis buffer. Samples were re-suspended in SDS sample buffer and analyzed by Western blotting. Measurement of cell viability Cell viability was assessed by the trypan blue staining assay. Ca9-22 cells were preincubated with wortmannin (Wort, 300 JPH203 in vivo nM) for 3 h or with actinomycin D (Act Cytidine deaminase D, 1 μg/ml ), cycloheximide (CHX, 1 μg/ml ), NF-κB inhibitor (PDTC, 5 μM), MAP kinase inhibitors, including a p38 inhibitor (SB203580, 5 μM), JNK inhibitor (SP600125, 1 μM) and ERK inhibitor (PD98059, 5 μM), at 37°C for 1 h and were then incubated with TNF-α for 3 h. Viability of the cells was determined by an exclusion test with trypan blue. Each measurement was repeated

three times independently. Those compounds were not toxic to the cells. (Additional file 2: Figure S1). Statistical analyses All experiments were performed in triplicate for each condition and repeated at least three times. Statistical analyses were performed using an unpaired Student’s t test. Multiple comparisons were performed by one-way analysis of variance and the Bonferroni or Dunn method, with results presented as the mean ± standard deviation. P-values less than 0.05 were considered statistically significant. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research B (to K.M.) and a Grant-in-Aid for Challenging Exploratory Research (to K.M.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Dr. Y.