Method: Caco-2 cells transfected with plasmid encoding CCL20-LUC plasmid are activated for 24h with the indicated concentrations of Flagellin (rec.) (AG-40B-0125) and (NLRC4 Mutant) (rec.) (AG-40B-0126). As a negative control, Flagellin (TLR5 Mutant) (rec.) (AG-40B-0127) is used. Luciferase assays are performed on cell lysates and luminescence is normalized to mock-treated cells.
Method: Mice WT are injected i.v. with 1µg of Flagellin (rec.) (AG-40B-0125)(=WT) or Flagellin (NLRC4 Mutant) (rec.) (AG-40B-0126). After 2 hours, levels of CCL20 in the serum are measured by ELISA. As control, WT mice are injected i.v. with 1µg of Flagellin (TLR5 Mutant) (rec.) (AG-40B-0127). PBS injected mice are used as negative control.
Method: Bone-marrow mouse macrophages (BMDMs) from WT or NLRC4 -/- mice are either left untreated, or incubated with streptolysin (SLO), or SLO with Flagellin (rec.) (AG-40B-0125) (=WT), or SLO with Flagellin (rec.) (TLR4 Mutant) (AG-40B-0126) at the indicated concentrations for 10min. Macrophages are then washed and incubated further for 2h before the cell extracts are prepared and immunoblotted for caspase-1 activation. p10 or p20 fragments are detected using the anti-Caspase-1 (p10) (mouse), mAb (Casper-2) (AG-20B-0044) or the anti-Caspase-1 (p20) (mouse), mAb (Casper-1) (AG-20B-0042), respectively.
The native flagellin [FliC] from Salmonella enterica Serovar Typhimurium strain ATCC14028 (aa 1-495) is fused at the N-terminus to a His-tag.
Detected by human and mouse TLR5 (Toll-like receptor 5) and mouse NLRC4 (Nod-like receptor [NLR] family caspase activation and recruitment domain [CARD] domain-containing protein 4).
Activation of TLR5 in human epithelial cell assays based on NF-κB luciferase fusions, in vivo on expression of CCL20 or IL-6. TLR5 can be stimulated in vitro by Flagellin (rec.) at concentration range of 5-50ng/ml and in vivo at a starting concentration of 0.1-1µg/mouse. Inflammasome NLRC4 can be activated at a concentration range of 1-5µg/ml on bone-marrow macrophages in the presence of streptolysin O (SLO, 25µg/ml), a protein that allows delivery of exogenous molecules into the cytosol of living cells.
|Endotoxin Content||<0.01EU/μg purified protein (LAL test).|
|Concentration||0.1mg/ml after reconstitution.|
|Reconstitution||Reconstitute with 100μl sterile water.|
|Formulation||Lyophilized. Contains PBS.|
|Other Product Data||
UniProt link P06179: Flagellin (Salmonella typhimurium)
|Shipping and Handling|
|Short Term Storage||+4°C|
|Long Term Storage||-20°C|
After reconstitution, prepare aliquots and store at -20°C.
Avoid freeze/thaw cycles.
Centrifuge lyophilized vial before opening and reconstitution.
|Use/Stability||Stable for at least 6 months after receipt when stored at -20°C.|
|Product Specification Sheet|
Flagellin is the subunit protein which polymerizes to form the filaments of bacterial flagella. It activates the innate immune system through the receptor Toll-like Receptor 5 (TLR5) and the intracellular protein NLRC4 (NLR family CARD domain-containing protein 4).
- Ezh2 competes with p53 to license lncRNA Neat1 transcription for inflammasome activation: J. Yuan, et al.; Nature Cell Death Diff. ahead of print (2022)