GPCR screening was performed according to the protocol reported by Chen et al.[36 (link)] Briefly, HTLA cells (a HEK293-derived cell line containing a stably integrated tTa-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin were seeded in 96-well tissue culture plates (Eppendorf) and transfected with 200 ng of S1PR4 plasmid (Addgene plasmid #66499)[25 (link)] for 20 h. The medium was then replaced with Dulbecco’s Modified Eagle Medium (DMEM) with 20 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer and 1% Penicillin/Streptomycin, and independently treated with compounds 1 and 2 , sphingosine-1-phosphate (S1P) (Fisher Scientific), and dimethyl sulfoxide (DMSO) solvent vehicle after 2 h. After 20 h of treatment, the luminescence was read by incubating each well for 20 min with 50 μl/well of Bright-Glo solution (Promega) diluted 20-fold in Dulbecco’s phosphate-buffered saline (DPBS) with 20 mM-HEPES. The luminescence was measured using Perkin Elmer EnVision 2100 plate reader.
S1pr4 plasmid
Manufactured by Addgene
The S1PR4 plasmid is a DNA construct that contains the coding sequence for the sphingosine 1-phosphate receptor 4 (S1PR4) gene. S1PR4 is a member of the G protein-coupled receptor family and plays a role in cellular signaling pathways. The plasmid can be used for the expression and study of the S1PR4 protein in various experimental systems.
Automatically generated - may contain errors
2 protocols using s1pr4 plasmid
1
GPCR Screening of S1PR4 Receptor
2
GPCR Screening of S1PR4 Receptor
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GPCR screening was performed according to the protocol reported by Chen et al.[36 (link)] Briefly, HTLA cells (a HEK293-derived cell line containing a stably integrated tTa-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin were seeded in 96-well tissue culture plates (Eppendorf) and transfected with 200 ng of S1PR4 plasmid (Addgene plasmid #66499)[25 (link)] for 20 h. The medium was then replaced with Dulbecco’s Modified Eagle Medium (DMEM) with 20 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer and 1% Penicillin/Streptomycin, and independently treated with compounds 1 and 2 , sphingosine-1-phosphate (S1P) (Fisher Scientific), and dimethyl sulfoxide (DMSO) solvent vehicle after 2 h. After 20 h of treatment, the luminescence was read by incubating each well for 20 min with 50 μl/well of Bright-Glo solution (Promega) diluted 20-fold in Dulbecco’s phosphate-buffered saline (DPBS) with 20 mM-HEPES. The luminescence was measured using Perkin Elmer EnVision 2100 plate reader.
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