NLRP3 Phospho-residue Mapping by Phospho Dot Blots.

When characterizing posttranslational modifications like phosphorylation, using efficient screening methods to map the phospho sites is essential, especially when dealing with large multi-domain proteins. NLRP3 (the NOD, LRR, and pyrin domain-containing protein 3), which initiates the formation of an NLRP3 inflammasome complex, is regulated posttranslationally by phosphorylation at several Ser and Tyr residues. However, determining sites of modification are not straightforward. For quick and reliable screening of the candidate phospho sites in NLRP3, we use a phospho dot blot assay which we describe here. This technique employs an in vitro kinase assay with a candidate kinase, Bruton's Tyrosine Kinase (BTK), and peptides derived from the region of interest in the protein that contains the potential phosphorylation sites. The reaction containing the phosphorylated peptides is quickly screened by a dot blot where the peptides are blotted with a commercially available anti-phospho-tyrosine antibody. This method can also be adapted to detect modified Ser or Thr residues and is an ideal screening assay to map phospho residues in NLRP3 or other proteins. This can be an initial screening procedure or can be complemented by other approaches such as site directed mutagenesis and by generating phospho site-specific antibodies.