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. 2020 Oct;98(4):462–474. doi: 10.1124/mol.120.119404

Fig. 7.

Fig. 7.

Comparing mRNA levels of endogenous Gα subunits and KOR signaling through Gαz and Gαi1 in both HEK 293T and CHO cells as measured with BRET. (A) Gα subunit mRNA profile in CHO and HEK 293 cells. Total mRNA was isolated from CHO and HEK 293 cell lines. Species-specific primers were used to determine relative mRNA levels to a GAPDH internal control. HEK 293 cells expressed the Gαz subunit transcript; CHO cells did not express detectable levels of Gαz. Gαo mRNA was not detected in either cell line. Data are the mean mRNA levels relative to GAPDH from three independent experiments performed in triplicate ± S.D. (B) U50,488 concentration-response curves were generated in HEK 293T and CHO cells after a 50-minute incubation. EC50 values were 7.3 ± 0.95 nM through Gαi1 and 5.0 ± 1.9 nM through Gαz in CHO cells compared with 5.6 ± 1.3 nM through Gαi1 and 1.5 ± 0.85 nM through Gαz in HEK 293T cells. (C) Concentration-response curves were generated for (-)pentazocine in HEK 293T and CHO cells after a 50-minute incubation. When the KOR was signaling through Gαi1, an Emax value of 31% ± 7.0% in CHO cells and of 35% ± 9.2% in HEK 293T cells (P = 0.57). KOR•Gαi1 signaling resulted in an EC50 value of 63 ± 24 nM in CHO cells and 110 ± 17 nM in HEK 293T cells (P = 0.023). (-)Pentazocine had similar Emax values when the KOR signaled through Gαz in both the CHO and HEK 293T cells (Emax values of 68% ± 6.6% and 79% ± 6.4%, respectively; P = 0.056). (-)Pentazocine had similar EC50 values when the KOR signaled through Gαz in CHO cells (EC50 value of 3.3 ± 1.1 nM) and HEK 293T cells (EC50 value of 7.3 ± 2.8 nM) (P = 0.051 between cell lines).