Cell culture and reagents. RPMI 8226, a human B cell line, was obtained from the American Type Culture Collection (Manassas, VA, USA), and maintained in an RPMI 1640 medium with 10% fetal bovine serum. We used trypan blue dye exclusion to assay the viability, which was typically greater than 95%. Peripheral blood was obtained from healthy volunteers with institutional review board approval. We isolated peripheral blood mononuclear
LAQ824 (PBMC) using density gradient centrifugation with Ficoll-Paque (Amersham Biosciences, USA). IKK-2 inhibitor BMS-345541 and stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) inhibitor SP 600125 were purchased from Calbiochem (San Diego, CA, USA). NF-κB inhibitor Pyrrolidine dithiocarbamate (PDTC), MAPK/ERK kinase (MEK) inhibitor PD 98059, and p38 inhibitor PD 169316 were purchased from A.G. Scientific, Inc (San Diego, CA, USA). When the inhibitors were used, the
cells were preincubated with SP 600125 for 10 min and with PDTC, BMS-345541, PD 98059, or PD 169316 for 1 h prior to stimulation with CpG-DNA. The following plasmids expressing mutant proteins were used for transfection: IκBαSR (IκBα super repressor) and MyD88 [22].
