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Figure 4 Constitutive activity of GPR3, GPR6, GPR12, and GPR12L(s) in chicken (A), duck (B), human (C), pig (D), and zebrafish (E). The activity of each receptor expressed in human embryonic kidney 293 (HEK293) cells was detected by dual-luciferase reporter assay. Each data point represents the mean ± SEM of 4 replicates (N = 4). ***, p < 0.001 vs. control (cells transfected with empty pcDNA3.1(+) vector). Western blot detection of the phosphorylated CREB (pCREB, (F)) and ERK (pERK, (H)) in HEK293 cells expressing cGPR6, cGPR12, and cGPR12L. pCREB and pERK levels were first normalized by that of actin and total ERK (tERK), respectively, and then expressed as the fold increase compared with the control group (transfected with empty pcDNA3.1(+) vector). ***, p < 0.001 vs. control. (G) The levels of pCREB increase dose-dependently in HEK293 cells transfected with increasing amount of cGPR6, cGPR12, or cGPR12L expression plasmid (50 ng, 100 ng, and 200 ng). Note. zebrafish has two GPR12L genes (GPR12La and GPR12Lb).