OVERVIEW
DOCUMENTS/REFERENCES
Biological Information
Background Information:
Several nuclear proteins have been implicated in the regulation of early T cell activation genes. Activator protein (AP-1), NF-kB, Oct-1, and AP-3 bind to sequences within the IL-2 or IL-2 receptor promoters essential for their transcriptional activation. The protein nuclear factor of activated T cells (NF-AT) binds to sequences within the IL-2 enhancer necessary for both inducibility and T cell-specific expression. NF-AT appears to be the product of an earlier gene in the T cell activation pathway and has been identified as a key element in the transcriptional regulation of the IL-2 gene.
Family:
Immune System
Sub Family:
Transcription Factor
Class:
NFAT
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 β-galactosidase gene is directed by the binding site for the NFAT-1 transcription factor, are used. Test compound or vehicle is preincubated with the cells for 20 minutes at 37°C, 5% CO2, then 1 μM A23187 and 100 ng/ml PMA are added for another 4 hours incubation. Test compound-induced β-galactosidase activity is determined by the conversion of FDG (fluorescein di-β-D-galactopyranoside) to fluorescein. Fluorescence intensity is measured with excitation at 485 nm and emission at 535 nm. Decrease of 50 percent or more (≥50%) in fluorescence intensity relative to vehicle control (0% inhibition), with 1 μM cyclosporin A defined as 100% inhibition, indicates significant inhibitory activity.
Incubation:
4 hr at 37°C
Control Inhibitor:
Cyclosporin A
Criteria For Significance:
≥ 50% Decrease in A23187 + PMA-induced beta-galactosidase expression relative to cyclosporine 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
Additional Information
Brand:
Panlabs
Testing Location:
Taiwan - Taipei
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