Figure 1

Validating the specificity of commercially available anti-NLRP3 antibodies by Western blotting. Nine commercially available anti-NLRP3 antibodies were tested in terms of their specificity against murine and human positive controls, including mouse spleen tissue, murine RAW 264.7 and human THP-1 macrophage cell lines. Antibody specificity was validated by testing protein expression in spleen tissue from Nlrp3 knockout mice as a negative control. RAW 264.7 cells were primed with (LPS 10 ng/mL) for 6 hours and were compared to vehicle untreated cells. THP-1 macrophages were primed with LPS (10 μg/mL) plus ATP (5 mM) for 3 hours and compared to vehicle control THP-1. 50 μg of total protein were loaded on a gel and blotted with anti-NLRP3 antibodies with the expected molecular weight at ~118 kDa. The positive control panel was blotted with the following anti-NLRP3 antibodies. (a) ProSci, 5447, (b) Novus 8N8E9, NBP2-03947, (c) abcam, 160971, (d) R&D Systems, MAB7578, (e) Novus NBP2-12446, (f) abcam, 91525, (g) Enzo ALX-804-819-C100, (h) Sigma HPA012878 and (i) Cell Signaling Technologies (D4D8T, 15101). Blots were exposed for 10 minutes. CST (15101) shows a specific band at the expected molecular weight for all positive controls, which is absent in spleen from Nlrp3 ^−/− sample. The response was amplified in stimulated RAW 264.7 and stimulated THP-1 cells compared to vehicle controls, demonstrating an upregulation of NLRP3 protein levels following inflammasome priming. In (j) duplicate samples of wild type and Nlrp3 ^−/− spleens were run in parallel, membrane was cut to probe with CST (15101) or Sigma (HPA012878) antibodies to verify their differences. An informative list of the antibodies is shown in Supplementary Table S1. Each blot was repeated in three biologically independent replicates (n = 3) and equal protein loading was confirmed by Coomassie Blue staining (Supplementary Fig. S2). WT: wild type, KO: knockout, VEH: vehicle.