After incubation for 30 min on ice, insoluble debris was removed by centrifugation for 15 min at 4��C. Total protein was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (GE Healthcare, Pittsburgh, PA, USA). The membranes www.selleckchem.com/products/baricitinib-ly3009104.html were then probed with antibodies against ��-tubulin (Thermo Fisher Scientific, Fremont, CA, USA), STAT3 (Cell Signaling Technology), or STAT3PY705 and visualized using SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific). Quantitative and Conventional Reverse-Transcription Polymerase Chain Reaction Assays Total RNA was isolated from colon tissues and cDNA was synthesized using a QuantiTech Reverse Transcription Kit (QIAGEN, Valencia, CA, USA), then mixed with QuantiFast SYBR Green polymerase chain reaction (PCR) master mix (QIAGEN) and specific primers.

Quantitative reverse-transcription PCR (qRT-PCR) was performed with an Applied Biosystems 7300 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) and analyzed by comparative Ct quantification [63]. The conventional PCR products were generated by AccuPower PCR Premix (Bioneer). The specific primers for genes used in this study were obtained from QIAGEN. Enzyme-linked Immunosorbent Assay (ELISA) Cell culture supernatants were analyzed for S100A9 levels using an S100A9 ELISA kit (Cyclex Co., Ltd, Nagano, Japan) according to the manufacturer’s instructions. Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) assays were performed using a Chromatin Immunoprecipitation Assay Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.

Briefly, Caco-2 cells were fixed with formaldehyde for 10 min at room temperature. After sonication (30 s on/1 min off for three cycles), the fragmented soluble chromatin was immunoprecipitated with anti-STAT3PY705 IgG or rabbit IgG. Cross-links were reversed by incubating at 65��C. Precipitated DNA was amplified by conventional PCR using specific primers covering the canonical STAT3 binding motif within the S100A9 promoter: forward, 5��-ACACATCTTGCCCACCAGGAGGCTA-3��; reverse, 5��-AAATCCTGCTCCCAGCAAGGGTTCC-3��. Statistical Analysis Data are presented as means �� standard deviations (SDs). P values <0.05 were considered statistically significant. Two-tailed Student's t-tests were conducted using the GraphPad Prism software (ver.

5.01; GraphPad Software, La Jolla, CA, USA). Supporting Information Figure S1 Changes in interleukin-6 (IL-6) expression in the colons of an experimental ulcerative colitis mouse model established by dextran sulfate sodium (DSS) treatment. (A) The difference (%) between baseline and subsequent body weight was measured daily for 11 days (left). Changes in clinical symptoms are Drug_discovery represented by the disease activity index (DAI; right). Data are presented as means �� standard deviations of values from six mice per group. *p<0.01, **p<0.001 vs.