Plasmids for ncAA incorporation (Appendix Fig S3)

Plasmid pIRE4‐BpaRS is a bicistronic construct built exactly as pIRE4‐Azi (Seidel et al, 2017; Serfling et al, 2018a). The backbone is originally based on pEGFP‐N1 (Clontech, Mountain View, CA) and bears a Kan/Neo resistance. The CMV‐EGFP sequence of pEGFP‐N1 was substituted with the AARS cassette, followed by the tRNA cassettes immediately downstream of the polyadenylation sequence. pIRE4‐Bpa contains a humanized gene of the E. coli BpaRS (Chin et al, 2003) (custom synthesized by GeneArt, Thermo Fisher Scientific) under control of a PGK promoter and four tandem repeats of a cassette for the expression of the tRNA suppressor from the Bacillus stearothermophilus tRNA^Tyr (BstYam), including a U6 promoter and a 5′‐trailer.

pNEU‐MmXYRS‐4xM15 is a bicistronic vector. The pNEU backbone is essentially the same as pcDNA3.1 with some variations in the restriction sites. The plasmid contains a humanized gene for the XYPylRS that recognizes BrEtY (Xiang et al, 2014) derived from Methanosarcina mazei PylRS (custom synthesized by GeneArt) under control of a CMV promoter, as well as four tandem repeats of the gene encoding for the enhanced pyrrolysine‐tRNA^M15 (Serfling et al, 2018a). The tRNA gene is depleted of the CCA end and is driven by a U6 promoter and followed by a T‐rich trailer.

HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM; high glucose 4.5 g/l, 4 mM glutamine, pyruvate; Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS; v/v; Thermo Fisher Scientific) and 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific) (full DMEM) at 37°C under 5% CO₂ and 95% humidity. Cells were passaged at ~ 70–80% confluence.

HEK293T cells were seeded at 500,000 cells per well in six‐well plates in 2 ml full DMEM. After 24 h, the media was exchanged with full DMEM supplemented with 250 μM p‐benzoyl‐phenylalanine (Bachem). Cells were transfected using PEI (Polysciences) at a ratio of PEI:DNA 3:1 (w/w) in lactate buffered saline (20 mM sodium lactate pH 4 and 150 mM NaCl) (Serfling & Coin, 2016; Serfling et al, 2018b). Cells were co‐transfected with three plasmids: (i) 900 ng of a plasmid bearing the arrestin stop codon mutant, (ii) 900 ng of pIRE4‐BpaRS, and (iii) 300 ng of a vector encoding the GPCR. Forty‐eight hours post‐transfection, the media was aspirated and exchanged with 1 ml activation buffer (PBS + 0.1% BSA). For the stimulation of the cells, the activation buffer was supplemented with 200 nM of the corresponding agonist (Ucn1 for CRF₁R, AVP for V₂R and PTH(1–34) for PTH1R). After 15 min at 37°C, the cells were irradiated with UV light for 15 min in a BLX‐365 crosslinker (Bio‐Budget Technologies, 365 nm; 4 × 8 W bulbs). Then, the activation buffer was aspirated and the cells were put at −80°C for 20–30 min, detached with 1 ml PBS supplemented with 1× protease inhibitor cocktail (Roche), and pelleted at 2,500 g for 10 min at 4°C. Cells were lysed in 80 μl Triton lysis buffer 1 (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X‐100, 1.5 mM MgCl₂, 1 mM EGTA, 1 mM DTT and 1× protease inhibitor) for 30 min on ice and vortexed every 10 min. The samples were centrifuged at 16,000 g for 10 min at 4°C, and supernatants were transferred to pre‐chilled tubes. For SDS–PAGE, 4 μl of supernatant was incubated for 30 min at 37°C in lithium dodecyl sulfate (LDS)‐sample buffer (250 mM Tris–HCl pH 8.5, 2% (w/v) LDS, 150 mM DTT, 0.4 mM EDTA, 10% (v/v) glycerol, and 0.2 mM Coomassie Brilliant Blue G). For deglycosylation, samples were treated with PNGaseF (New England Biolabs) according to the manufacturer's protocol and LDS‐sample buffer was added before SDS–PAGE.