pJAP2

Protein Expression Eukaryotic cells - HEK293 AT1R

Experiment
Protein Expression Eukaryotic cells - HEK293 AT1R
Product
pJAP2 from Christopher G. Tate, MRC Laboratory of Molecular Biology, Cambri
Manufacturer
Christopher G. Tate, MRC Laboratory of Molecular Biology, Cambri

Protocol tips

Protocol tips
transiently transfected (GeneJuice, Novagen) into adherent mammalian iHEK cells or iGnTI− cells following the manufacturer's protocol. Cells were grown in Dulbecco's modified Eagle's media supplemented with 10% tetracycline-free foetal bovine serum (Invitrogen) and 5 μg/ml blasticidin (Invitrogen) and incubated at 37 °C in an atmosphere containing 5% CO2. Expression of plasmids was induced by addition of 1 μg/ml tetracycline and incubated at 37 °C for 24 h.

Publication protocol

Mammalian expression plasmids for the expression of AT1R (pJAP2), A1R (pJAP34) and A1R-GL26 (pJAP37) were amplified in E. coli strain DH5α, purified using a Maxi-prep kit (Qiagen) and transiently transfected (GeneJuice, Novagen) into adherent mammalian iHEK cells or iGnTI− cells following the manufacturer's protocol. Cells were grown in Dulbecco's modified Eagle's media supplemented with 10% tetracycline-free foetal bovine serum (Invitrogen) and 5 μg/ml blasticidin (Invitrogen) and incubated at 37 °C in an atmosphere containing 5% CO2. Expression of plasmids was induced by addition of 1 μg/ml tetracycline and incubated at 37 °C for 24 h. Stable cell lines were generated by selection with media containing 200 μg/ml Zeocin (Invitrogen). An iGnTI− stable cell line expressing a thermostable mutant of SERT, SERT-SAH9 (J. Andréll and C. Tate, unpublished results; Ref. [38]) and (iGnTI− SERT-SAH9-GFP-H10) was kindly provided by J. Andréll. A highly expressing clonal AT1R-GFP-H10 cell line was selected from a polyclonal cell line using fluorescence-activated cell sorting. After expression, cells were washed twice in phosphate-buffered saline (PBS), counted using the Countess Automated Cell Counter (Invitrogen), pelleted (1200g for 5 min) and resuspended at 10 million cells per millilitre in ice-cold cell buffer [50 mM Tris (pH 7.4) and 150 mM NaCl supplemented with Complete EDTA (ethylenediaminetetraacetic acid)-Free Protease Inhibitor Cocktail (Roche)]. Cell suspensions were flash frozen in liquid nitrogen and stored at − 80 °C.

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