NLRP3

NLRP3 (NACHT, LRR and PYD domains-containing protein 3) is a protein encoded by the NLRP3 gene. NLRP3 is expressed predominantly in macrophages and plays a crucial role in innate immunity and inflammation.

Overview

​Protein function, expression, and isoforms

Protein function 

  • NLRP3 may function as an inducer of apoptosis.
  • It interacts selectively with ASC and this complex may function as an upstream activator of NF-kappa-B signaling. 
  • It inhibits TNF-alpha induced activation & nuclear translocation of RELA/NF-KB p65, and also inhibits transcriptional activity of RELA. 
  • NLRP3 activates caspase-1 in response to a lot of triggers including bacterial or viral infection which leads to processing and release of IL1B and IL18.

Expression

  • NLRP3 is expressed in blood leukocytes and is also strongly expressed in polymorphonuclear cells and osteoblasts. 
  • NLRP3 is undetectable or expressed at a lower magnitude in B- and T-lymphoblasts, respectively. 
  • High levels of NLRP3 expression are detected in chondrocytes
  • NLRP3 is also detected in non-keratinizing epithelia of oropharynx, esophagus and ectocervix and in the urothelial layer of the bladder

Isoforms

  • Human: Isoform 1: 84 kD (predicted)
  • Human: Isoform 2: 106 kD (predicted)
  • Human: Isoform 3-6: 110-120 kD (predicted)
  • Mouse: Isoform 1: 96 kD (predicted)
  • Mouse: Isoform 2-4: 110-120 kD (predicted)
  • Rat: Isoform 1: 119 kD (predicted)

​Applications

NLRP3 can be challenging target to work with in western blot. 

Western blot

NLRP3 is regulated by post-translational modifications, such as ubiquitination and phosphorylation. It has several isoforms in human and mouse. Therefore, it’s may exist several bands in a western blot assay. Moreover, the predicted molecular weight of NLRP3 is about 118 kDa. The 8% gel is more appropriate. Large proteins (>100 kDa) will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will facilitate transfer. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also prevent precipitation. The observed band size of NLRP3 may not be the same as predicted MWs in western blot due to the different forms of the target.



Sample preparationAdd adequate protease inhibitors (or phosphatase inhibitors for proteins modified by phosphorylation) to avoid target protein degradation

Ultrasonicate samples to enrich more target proteins

Keep samples on ice during the whole WB process

Perform a Bradford assay, a Lowry assay or a bicinchoninic acid (BCA) assay to determine the protein concentration.
ElectrophoresisFor large proteins (the MW of target protein >100 kDa), be sure to run samples in 8% or lower separating gel.
TransferringIt is preferred to add SDS to a final concentration of 0.1% in the transfer buffer for large proteins.

Wash PVDF membrane to remove methanol completely.

To determine if the transfer is successful by visualization of proteins in membranes using Ponceau S.
Maximizing signalThe level of NLRP3 expression can be elevated when cells are treated with LPS1

There may be multiple bands of NLRP3 in WB assay. NLRP3 can be regulated at post-translational level, such as phosphorylation, ubiquitination. And NLRP3 has 6 isoforms in human(predicted).
ControlsPositive:
Human THP1 WT cells treated with 200 ng/ml LPS for 3h
Rat spleen lysate
Negative:
Jurkat (human t cell leukaemia t lymphocyte) whole cell lysate

Find full information on working with NLRP3: 

Find full information on working with NLRP3: 
In English (link to pdf) 
In Mandarin (Link to pdf) 

References

  1. Qiao, Y., Wang, P., Qi, J., Zhang, L., Gao, C. TLR-induced NF-κB activation regulates NLRP3 expression in murine macrophages. FEBS Lett. 586, 1022-6 (2012)