MSU activates NLRP-3 without any effect on the NF-κB pathway. (A) Expression levels of NLRP-3 during MSU stimulation. Confluent cells were stimulated with 0.5 mg MSU for the indicated times and then subjected to immunoblot analysis by using anti-NLRP-3 and anti-β-actin antibodies. Macrophages (MΦ) stimulated for 24 hours with LPS, 100 ng/ml, were used as positive control. (B) Quantification of NLRP-3 levels in MSU-stimulated OBs. Pixel-density results were normalized with β-actin, and cumulative data are expressed as mean ± SEM (three different donors). Statistical analysis was performed by using the one-way ANOVA Bonferroni multiple-comparison test; means without a common letter differ: P < 0.05. (C) Analysis of the NF-κB pathway activation. Confluent OBs were stimulated with 0.5 mg MSU for the indicated times. Protein lysates were subjected to immunoblot analysis for phosphorylated IκB (P-IκB) or IκB. As a positive control for IκB phosphorylation, OBs were stimulated with TNF-α (50 ng/ml) for the indicated times.