Plasmids encoding N-terminally FLAG-tagged β2AR and HA-tagged PTHR1 were described previously (39) and used for the generation of HA-tagged PTHR1 point mutants H 2.50 R, T 6.42 P and I 7.56 R using the Geneart Site-directed Mutagenesis Kit (Thermo Fisher Scientific). Plasmid encoding wild-type TBXA2R was kindly provided by A. Inoue (Tohoku University, Sendai, Japan). Wildtype H3R, GPR3, GPR6, GPR12 and G protein subunits were either purchased from cDNA.org in pcDNA vectors or amplified from previously described G protein sensors (34) . PcDNA-Gαo1-Nluc, pcDNA-Gα13-Nluc and pcDNA-Gα15-Nluc were synthesized by GenScript. The subunits were then fused to cpVenus 173 or Nluc at the indicated positions (Fig. 1c) and encoded on a tricistronic T2A-IRES vector as described previously (32, 34) using established PCR techniques and restriction enzymes. All constructs were verified by sequencing (Eurofins genomics).
Pcdna gα13 nluc
Manufactured by GenScript
PcDNA-Gα13-Nluc is a lab equipment product designed for genetic engineering and cell biology research. It is a plasmid vector that contains the Gα13 subunit of the G protein-coupled receptor (GPCR) family fused with the Nanoluciferase (Nluc) reporter gene. This product can be used for studying GPCR signaling pathways and protein-protein interactions in cellular systems.
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2 protocols using pcdna gα13 nluc
1
Generation of GPCR Receptor Mutants
2
Generation of GPCR Receptor Mutants
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Plasmids encoding N-terminally FLAG-tagged β2AR and HA-tagged PTHR1 were described previously (39) and used for the generation of HA-tagged PTHR1 point mutants H 2.50 R, T 6.42 P and I 7.56 R using the Geneart Site-directed Mutagenesis Kit (Thermo Fisher Scientific). Plasmid encoding wild-type TBXA2R was kindly provided by A. Inoue (Tohoku University, Sendai, Japan). Wildtype H3R, GPR3, GPR6, GPR12 and G protein subunits were either purchased from cDNA.org in pcDNA vectors or amplified from previously described G protein sensors (34) . PcDNA-Gαo1-Nluc, pcDNA-Gα13-Nluc and pcDNA-Gα15-Nluc were synthesized by GenScript. The subunits were then fused to cpVenus 173 or Nluc at the indicated positions (Fig. 1c) and encoded on a tricistronic T2A-IRES vector as described previously (32, 34) using established PCR techniques and restriction enzymes. All constructs were verified by sequencing (Eurofins genomics).
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