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. 2021 Mar 5;296:100503. doi: 10.1016/j.jbc.2021.100503

Figure 4.

Figure 4

Generation of stable cell lines using a MeNArC polycistronic vector for use in high-throughput screening.A, schematic representation of the polycistronic MeNArC vector containing the coding region for the ArC and MeN components separated by a P2A ribosomal slippage sequence that allows for the expression of both of the proteins from a single vector. The vector was stably integrated into CHO cells expressing each of the opioid receptors to carry out the MeNArC assay. B, dose–response curves of the MeNArC assay for the MOR, KOR, DOR, and NOP cells showing increase in luminescence (au) with increasing concentration of agonist; KOR (pEC50: −7.86 ± 0.10), MOR (pEC50: −7.7 ± 0.15), DOR (pEC50: −8.63 ± 0.12), or NOP (pEC50: −8.15 ± 0.24), stably transfected into CHO-K1 cells together with the β-arrestin2 version of the polycistronic ArC-P2A-MeN plasmid. C, β-arrestin2 recruitment tested with the MOR partial agonists morphine and buprenorphine normalized to the full agonist DAMGO. D and E, representative Z’-factor data from one data set conducted with (D) MOR and (E) DOR on a 384-well plate in the absence (gray) or presence of agonist (blue). F, comparison of Z’-factor data compiled from three individual experiments showing mean values (red middle line) of 0.67 ± 0.06 and 0.69 ± 0.09 for MOR and KOR, respectively, and SD. The open circles represent the values determined from the plots shown in (D and E).