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 stripped and probed with anti-β-actin antibodies as a loading control. GST-R5BD pull-down assay The GST-R5BD pull-down assay RGFP966 supplier 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, ARN-509 clinical trial 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 check details nM) for 3 h or with actinomycin D (Act Adenosine 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.