| Solution Information | help | |
| Enzyme: | Sphingosine 1-phosphate receptor 3 | |
| inhibitor: | BDBM62228 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: In this assay, a CHO cell line containing human S1P3 and the beta-lactamase (BLA) reporter-gene under control of the nuclear factor of activated T-cells (NFAT) promoter was used to measure S1P3 antagonism by test compound. Stimulation of S1P3 by S1P induces transcription of NFAT-BLA via a G-alpha16 protein coupled signaling cascade, and an increase in BLA activity. BLA activity is measured using a fluorescent BLA substrate. As designed, a compound that acts as a S1P3 antagonist will decrease NFAT-BLA transcription and decrease well fluorescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 25 micromolar. Protocol Summary: Cells were cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) containing 10% v/v heat inactivated bovine growth serum, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, 5 mM L-G | |
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