Fig. 3: Interaction of GPRC5B with other prostanoid receptors.

A Western blot detection of HA and FLAG signals in lysates of HEK cells expressing FLAG-tagged GPRC5B in combination with different HA-tagged prostanoid receptors before (“input”) and after immunoprecipitation of GPRC5B-FLAG/Myc (“Pulldown FLAG”). B Expression of prostanoid receptors was determined by RNA sequencing (n = 12) (same samples as in Fig. 1A). C Calcium mobilization induced by EP1 agonist 17-pt-PGE₂ in HEK cells transfected with calcium sensor aequorin, EP1 receptor, and control siRNA (siContr) or siRNA directed against GPRC5B (siG5B), respectively (n = 8). RLU: relative light unit. cAMP production induced by EP2 agonist butaprost (D) or EP4 agonist L-902688 (E) was determined in HEK cells transfected with a cAMP GloSensor plasmid, EP2 (D) or EP4 (E) receptors, as well as control siRNA or siRNA directed against GPRC5B, respectively (n = 8). F, G cAMP production induced by EP2 agonist butaprost (F) or EP4 agonist L-902688 (G) in HEK cells transfected with a cAMP GloSensor plasmid, EP2 (F) or EP4 (G) receptors, as well as empty vector (EV) or GPRC5B (G5B) as indicated (n = 8 and 4, respectively). H Butaprost-induced cAMP production in HEK cells transfected with cAMP GloSensor plasmid and empty vector (EV) or GPRC5B as indicated (without EP2 transfection; n = 4). I, J Butaprost-induced cAMP production in HEK cells transfected with empty vector (EV) or GPRC5B in the presence (I) or absence (J) of overexpressed EP2 receptor was determined by ELISA (n = 6/8/7/7/6/8/6/7 in I, 6(J)). K Spatial correlation between HA and FLAG signals in HEK cells co-transfected with HA-EP2 and GPRC5B-FLAG/Myc: exemplary photomicrographs and statistical analysis (Pearson’s coefficient, n = 29 cells). Data are means ± SEM; comparisons between genotypes (C-E) or both genotypes and treatments (G-J) were done using two-way ANOVA and Sidak’s post hoc test (C-E, G-J) or one-way ANOVA with Tukey’s multiple comparison test (F). EV, empty vector; G5B, GPRC5B-encoding plasmid; n, number of independent experiments; siContr, scrambled control siRNA; siG5b, siRNA directed against GPRC5B; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are provided as a Source Data file.