pJAP34

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	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.

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 An iGnTI− stable cell line expressing a thermostable mutant of SERT, SERT-SAH9 (J. Andréll and C. Tate, unpublished results; Ref.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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