NLRP3/NALP3 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

NACHT, LRR and PYD domains-containing protein 3 (NALP3), Nucleotide-Binding Oligomerization Domain, or Leucine Rich Repeat and Pyrin Domain Containing 3 (NLRP3) (human NLRP3-isoform 2 theoretical molecular weight 118kDa) acts as a cytosolic receptor for stress-signals initiated by a wide variety of pathogen-associated molecular patterns (PAMPS) and danger-associated molecular patterns (DAMPs) (1). NLRP3 belongs to the family of NOD-like receptors, a type of pattern-recognition receptor (PRR) which participates in innate immune responses. The exact mechanisms leading to NLRP3 activation are still not fully resolved. Some proposed mechanisms for NLRP3 inflammasome activation include induced ion channel flux as exemplified by potassium efflux, sodium influx and calcium signaling, generation of mitochondrial reactive oxygen species (ROS), and lysosomal destabilization (2). Activation of NLRP3 induces its association with the adaptor protein Apoptosis-associated Speck-like protein containing CARD (ASC) forming a complex that binds to Caspase-1 (1, 2). The inflammasome complex, comprised of NLRP3, ASC and Caspase 1, induces the processing of pro-IL-1beta and pro-IL-18 and release of the mature inflammatory cytokines IL-1beta and IL-18 and pyroptosis (1-3).

NLRP3 is expressed in a variety of cell types such as monocytes, dendritic cells, lymphocytes and epithelial cells (3). Abnormal NLRP3 activation may occur as the result of inherited mutations and is associated with diseases such as hereditary periodic fevers (HPFs) and familial cold autoinflammatory syndrome (FCAS). Additionally, abnormal NLRP3 activation is associated with a variety of diseases such as gout, obesity, atherosclerosis, Alzheimers, multiple sclerosis and type 2 diabetes (1, 3). NLRP3 inflammasome is regulated by several post-translational modifications (e.g., ubiquitination, phosphorylation and sumoylation) as well as by various interacting proteins (e.g., JNK1, FBXO3, TXNIP, MARK4 and PKD) (4).

References

1. Abderrazak, A., Syrovets, T., Couchie, D., El Hadri, K., Friguet, B., Simmet, T., & Rouis, M. (2015). NLRP3 inflammasome: From a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases. Redox Biology. https://doi.org/10.1016/j.redox.2015.01.008

2. Yang, Y., Wang, H., Kouadir, M., Song, H., & Shi, F. (2019). Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors. Cell Death and Disease. https://doi.org/10.1038/s41419-019-1413-8

3. Zahid, A., Li, B., Kombe, A. J. K., Jin, T., & Tao, J. (2019). Pharmacological inhibitors of the nlrp3 inflammasome. Frontiers in Immunology. https://doi.org/10.3389/fimmu.2019.02538

4. Kelley, N., Jeltema, D., Duan, Y., & He, Y. (2019). The NLRP3 inflammasome: An overview of mechanisms of activation and regulation. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms20133328

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-13675

• NLRP3/NALP3 Antibodies
• NLRP3/NALP3 ELISA Kit
• NLRP3/NALP3 Lysates
• NLRP3/NALP3 Proteins