Fig. 4. HA stimulates Ca²⁺ mobilisation in CHO-K1 cells stably transfected with GPR37-FLAG, G16 and apoaequorin. (A) The Ca²⁺-bioluminescence response was measured at 469 nm and is expressed in relative light units (RLU), from which the medium response was subtracted. Values are given as means ± s.d. CHO-G16-AEQ cells stably expressing GPR37-FLAG (CHO-GPR37) responded with an EC₅₀ value of 3.3 nM; the endogenous response of CHO-G16-AEQ cells (CHO) resulted in an EC₅₀ value of 11 nM. Data show representative results of three independent experiments. (B) CHO-GPR37 (upper panel) and CHO cells (lower panel) reacted with anti-GPR37(R1) antibody [-GPR37(R1)], a polyclonal antiserum produced against the conserved intracellular C-tail. (C) Western blot analysis of membrane fractions confirmed an increased expression of GPR37 in transfected cells. The mouse hippocampal cell line HT22, expressing GPR37 endogenously, was used as a positive control.