All chemicals used in this study Epigenetics inhibitor were purchased from Sigma Chemical Co. (St Louis, MO, USA) unless otherwise specified. Primary

antibodies specific to p-ERK (sc-7383), p-JNK1/2 (sc-6254), NF-κB subfamilies (p50, sc-8414; p52, sc-7386; p65, sc-8008; RelB, sc-226, c-Rel, sc-6955) and Skp2 (sc-7164) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies specific to p-p38 MAP kinase (#9215, Cell Signaling Technology, Danvers, MA, USA), I-Ad MHC class II molecule (553611, BD Bioscience, Franklin Lakes, NJ, USA), p27kip (14-6716-81, eBioscience, San Diego, CA, USA), IL-16 (MAB1727, R&D systems, Minneapolis, MN, USA) and tubulin (T3526, Sigma Chemical Co.) were purchased from the indicated suppliers. NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, buy AZD6244 IL, USA) were used to separate and prepare cytoplasmic and nuclear extracts from the cells. Anti-I-Ad antibody was purified from serum-free culture supernatant obtained from the MK-D6 hybridoma cell line [20]. The resting B cell line, 38B9, established from BALB/c mice (d-haplotype), was cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS, PAA, Etobicoke, Ontario, Canada) and 50 μm 2-mercaptoethanol at 37 °C in a humidified 5% CO2 incubator. Cells (1.5 × 107 38B9) were washed with cold PBS, lysed in lysis buffer [1% Triton X-100 in 50 mm Tris–HCl pH 7.4, 150 mm NaCl,

1 mm Na3VO4, 5 mm NaF and protease inhibitor cocktail (Complete, STK38 Mini; Roche Applied Science, Mannheim, Germany)] for 30 min

at 4 °C, and subsequently centrifuged for 10 min at 13 000× g. Protein concentration was determined using a commercial Bradford assay system with bovine serum albumin (BSA) as a standard. After treatment with 5× Laemmli reducing sample buffer, the lysates were resolved by 12% SDS-PAGE and transferred electronically to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% BSA/TBST and incubated first with specific antibodies and then with a horseradish peroxidase-conjugated secondary antibody. Finally, the blots were developed using the ECL detection system (Pierce Biotechnology). Cell lysates were precleared by incubating them with a slurry of protein A-sepharose 4B (GE Healthcare Bio-Science, Piscataway, NJ, USA) with rotation for 30 min at 4 °C. The samples were then centrifuged at 5000 g for 3 min, and the pellets were discarded. The collected supernatants were mixed and rotated with MK-D6 anti-I-Ad antibody for 4 h at 4 °C. A slurry of protein A-sepharose 4B was added, and the mixture was incubated overnight at 4 °C. The beads were then washed five times with lysis buffer before they were resuspended in sample reducing buffer. 38B9 resting B cells (5 × 106) that were untreated or treated with LPS (50 μg/ml) or LPS together with MK-D6 anti-I-Ad antibody (50 μg/ml) were lysed in lysis buffer.