Pegfp n1 mammalian expression vector

The HaloTag7 (Promega) coding sequence was amplified by PCR, and was fused at the C-terminus of mouse mGluR3 with an In-Fusion HD Cloning Kit (Clontech). To quantify the expression of wild type and HaloTag-fused mGluR3 by Western blotting analysis, the epitope sequence of the anti-bovine rhodopsin monoclonal antibody Rho1D4 was also fused at the C-terminus. The cDNAs of mGluR3s were introduced into the pcDNA3.1 mammalian expression vector (Invitrogen). The cDNAs of other GPCRs (ADRB2, HTR2A, HRH1, ADORA2A, FFAR4, CXCR4, F2R, GCGR) were purchased from Promega, and the receptor coding sequences were inserted into pFC14K HaloTag CMV Flexi Vector. The CD86 (M1-R277) coding sequence was amplified by PCR, and inserted into the pEGFP-N1 mammalian expression vector (Clontech), where the coding sequence of EGFP was swapped with that of HaloTag7. The SNAP-tag (NEB) coding sequence was amplified by PCR and inserted in the loop region between L91 and G92 of the mouse Gα o subunit coding cDNA, as reported previously (12) . The SNAP-tagged Gα o subunit coding sequence was inserted into the pFC15A HaloTag CMVd1 Flexi Vector without the HaloTag coding sequence to reduce the expression level for single-molecule imaging. The cDNA of GFP-tagged CLC was constructed as previously reported (45) (link).