Complete protease inhibitors (Roche). Protein concentrations in lysates were determined using a DC Protein Assay (BioRad) to ensure that equal amounts of lysate protein were used for immunoprecipitation. Protein A/G Plus agarose beads

GEArray TM analysis The non-radioactive GEArray TM original and Q series cDNA expression array filters (Original Series Mouse Common Cytokine-1 Gene Array, Cat. No.: mGEA101207N and Mouse Inflammatory Cytokine/Receptor Gene Array Q series, Cat. No.: MM-002N-4) were used, and hybridization procedures were as described by the manufacturer (SuperArray Inc., Bethesda, MD). Total RNA was isolated from tissue samples of mice after in vivo mAb treatment using the RNeasy Midi Kit (Qiagen). The quality of RNA was assessed using an Agilent Technologies 2100 Bioanalyzer (Caliper Technologies Corp.). Aliquots of total RNA (5 g) were taken to prepare biotin dUTP-labeled cDNA probes using gene specific primers and 200 U of Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Invitrogen). The array filters were hybridized (60 o C, 17 h) with biotin-labeled probes, washed twice with 2x saline sodium citrate buffer (SSC)/1% SDS and then twice with 0.1 x SSC/1% SDS at 60oC for 16 min each. The arrays were then developed using alkaline phosphatase-conjugated streptavidin and CDP-Star substrate (SuperArray Inc.), and visualized by autoradiography. The relative amount of each transcript was determined by scanning densitometry, and these signal intensities were compared to GAPDH (Original Series Mouse Common Cytokine-1 Gene Array) and the cyclophilin A signal (Mouse Inflammatory Cytokine/Receptor Gene Array Q series). For Gene Array Q series analysis, data was analyzed (pUC18 plasmid DNA background subtraction and normalization to cyclophilin A) using GEArray Analyzer Software, as described by the manufacturer (www.superarray.com). All cDNA array experiments were performed twice. A greater than two-fold change in signal intensity was considered significant.