Fig. 3

Detection of ecto-GPR37 by the NanoLuc (NL)-based immunoassay. a Schematic representation showing the putative metalloproteinase (scissors)-mediated cleavage and shedding of the GPR37 N-terminal domain (i.e. ecto-GPR37) in a GPR37 construct containing the HA (filled red circle) and NL tags at its N-terminus (i.e. ecto-GPR37^NL). Upon expression in HEK-293 cells, the GPR37^NL ectodomain was released by shedding and detected in the culture medium by monitoring its NL enzymatic activity after incubation of the growth medium of cells transfected with mock (Veh) or mouse (b)/human (c) GPR37^NL with coelenterazine 400a and determination of the luminescence (RLU, relative light units). d Assessment of ecto-GPR37 in 1 μl, 3 μl and 5 μl of cerebrospinal fluid of GPR37^+/+ and GPR37^−/− mice. The purified mouse ecto-GPR37^NL was used as a tracer in the competitive ELISA. Results are expressed as RLU (mean ± SD, n = 3). Dashed line represents the RLU_max (i.e. RLU in the absence of CSF). *P < 0.05 and **P < 0.01 vs RLU_max, one-way ANOVA followed by Dunnett’s post-hoc test. e Assessment of ecto-GPR37 in post-mortem CSF from NC (n = 8) and PD (n = 8) subjects from Bellvitge Hospital using the GPR37-NL-based ELISA assay. **P < 0.01, Student’s t-test