OVERVIEW
DOCUMENTS/REFERENCES
Biological Information
Background Information:
In resting cells, cytoplasmic location of the nuclear transcription factor NF-kB is bound by an inhibitory subunit IkB; binding of IkB effectively masks the nuclear localization sequences present on the P50 and P65 subunits of NF-kB, preventing nuclear translocation. It appears that upon cellular stimulation, a signal transduction pathway is activated, leading to phosphorylation of key serine residues in the IkB polypeptide, whereupon the NF-kB-IkB complex dissociates, IkB is rapidly degraded, and the unmasked nuclear localization signal allows NF-kB to translocate into nuclei and activate the transcription of specific genes. It is known that NF-kB regulates many pro-inflammatory and prothrombic factors produced by activated leukocytes. NF-kB represents a master regulator of inflammation and is, therefore, an attractive target for drug development. NF-kB may also facilitate replication of HIV-1.
Family:
Immune System
Sub Family:
Transcription Factor
Class:
RHR
Organism:
Human
Construct Details:
Endogenous
Assay Information
Assay Type:
Functional
Assay Sub Type:
Cell Based
Functional Mode:
Antagonist
Detection Method:
Spectrofluorimetry
Measured Response:
Fluorescence
Testing Information
Procedure Summary:
Human T lymphocytic Jurkat cells, transfected with a response element-lacZ reporter in which transcription of the b-galactosidase gene is directed by the binding site for the NF-kB transcription factor, are used. Test compound and/or vehicle is preincubated with the cells (2 x 10⁶ cells/ml) for 20 minutes in RPMI-1640 buffer pH 7.4 at 37°C, then 0.5 µM A23187 and 50 ng/ml PMA are added for another 4 hours incubation period. Test compound-induced b-galactosidase activity is determined by the conversion of FDG (fluorescein di-b-D-galactopyranoside) to fluorescein. Fluorescence intensity is read on a SpectroFluor Plus plate reader. A decrease of 50 percent or more (≥50%) in fluorescence intensity, relative to 10 µM cyclosporin A, indicates significant inhibitory activity. These same concentrations are concurrently applied to a separate group of treated cells and evaluated for possible compound-induced cytotoxicity only if significant stimulation or inhibition is observed (Cat. #361100).
Incubation:
4 hr at 37°C
Control Inhibitor:
Cyclosporin A
Criteria For Significance:
≥ 50% Decrease in A23187 + PMA-induced beta-galactosidase expression relative to cyclosporin A
Test Concentration / Dose:
Compounds are screened at 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0.003 μM
Test Sample Requirements:
75 μl of 10 mM stock or 1 mg (pre-weighed) powder (assuming FW ≦500 and purity 100%)
Minimum Order Quantity:
16
Turnaround Time
Standard:
20 Business Days
Reference Compound Data
Clinical Relevance
Therapeutic Area:
Inflammation/Allergy
Additional Information
Brand:
LeadHunter
Testing Location:
Taiwan - Taipei
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