Anti phospho ikbα

Cellular lysates were prepared and analyzed by Western Blotting as previously described [86 (link)]. Proteins were extracted by incubating the cells in lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 2 mM EGTA; 2 mM EDTA; 25 mM NaF; 25 mM β-glycerophosphate; 0.1 mM NaV; 0.2% Triton X-100; 0.3% Nonidet P40; proteinase inhibitor, Roche, Basel, Switzerland). Following centrifugation, protein concentrations of supernatants were determined by the BCA™ Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Next, 30–50 μg of protein per sample was separated electrophoretically using 10% SDS–PAGE gels and blotted onto Hybond™-C-Extra Nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). Proteins of interest were detected using the following antibodies: anti-phospho-IkBα (Ser32/36, mouse, monoclonal, 5A5, Cell Signalling, Danvers, MA, USA), anti-IkBα (rabbit, polyclonal, C-21, Santa Cruz, Singapore), anti-PCNA (mouse, monoclonal, PC10, Abcam), and anti-ubiquityl-PCNA (Lys164, rabbit, monoclonal, D5C7P, Cell Signaling). Chemiluminescence signals were detected on a ChemiDocMP System (BioRad, Hercules, CA, USA) using Clarity^TM Western ECL Substrate (BioRad) and Band intensities were quantified using ImageLab software 4.1. Intensity values of the protein of interest were normalized to the values of the corresponding loading control.

Red protein G affinity beads, RIPA buffer, and protease inhibitor cocktail were purchased from Sigma-Aldrich Corporation (St. Louis, MO). The HSP90 inhibitors, AUY-922 and AT13387, were obtained from Selleck Chemicals (Houston, TX). The BCA Protein Assay Kit was purchased from Pierce Co. (Rockford, IL), and Western blot membranes were purchased from GE Healthcare (Chicago, IL). All antibodies used in Western blots and immunoprecipitation have published immunospecificity data available online. Rabbit anti-AKT, anti-phospho AKT, anti-IKBα, anti-phospho IKBα, anti-STAT3, anti-phospho STAT3, anti-VE-cadherin, anti-occludin, anti-cofilin, and anti-phospho cofilin were purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal anti-β-actin and anti-p53 (P8999) were purchased from Sigma-Aldrich Corporation, and secondary IRDye 800CW goat anti-rabbit (926-32211) and IRDye 680RD goat anti-mouse (926-68070) were purchased from LI-COR Biosciences (Lincoln, NE). For SDS-PAGE, Protogel (30% acrylamide mix) and TEMED were purchased from National Diagnostics (Atlanta, GA), Tris-HCl buffer was purchased from Teknova (Hollister, CA), 10% SDS and ammonium persulfate were purchased from Thermo Fisher Scientific, and protein dual-color standards and tricine sample buffer were purchased from Bio-Rad Laboratories.

RAW264.7 cells were seeded into TC-treated six-well tissue culture plates (1 × 10⁶ cells/well) and incubated at 37°C under 5% CO₂. Twelve hours later, cells were treated with DMEM media alone (negative control), DMEM with 100 ng/ml LPS (positive control), or DMEM with C. butyricum S-45-5-Cell (1 × 10⁶ CFU/ml) or S-45-5-Sup (1 × 10⁶ CFU/ml), and the cells were collected at 0, 8, 12, and 16 hpt. After washing the pellets with PBS, whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies indicated: anti-IRF3 (Cell Signaling, 4302S), anti-phospho-IRF3 (Ser396) (Cell Signaling, 4947S), anti-STAT1 (Cell Signaling, 9172), anti-phospho-STAT1 (Tyr701) (Cell Signaling, 9167S), anti-p65 (Cell Signaling, 4764S), anti-phospho-p65 (Cell Signaling, 3031S), anti-IkBα (Cell Signaling, 9242S), anti-phospho-IkBα (Cell Signaling, 2859S), or anti-β-actin (Santa Cruz, SC#47778) antibodies. A Las-3000 miniLumino Image Analyzer (ECL-GE Healthcare, UK) equipped with a Chemiluminescence Detection System (ECL-GE Healthcare, UK) was used to visualize the respective proteins using a horseradish peroxidase-conjugated secondary antibody (Sigma, USA).