Fig. 3

V2Rp4 is a functional receptor for ESP22. a Dual-color ISH staining of a VNO section from an ESP22-stimulated female mouse labeled with the Egr1 cRNA probe (green) and V2Rp clade-specific cRNA probe (magenta). Closed arrowheads show double-labeled cells. Scale bar, 50 µm. b Percentage of V2R-positive VSNs among ESP22-induced Egr1-expressing neurons. c Dual-color ISH staining with Egr1 (green) and each V2Rp cRNA probe (magenta). Open arrowheads show Egr1-positive cells; closed arrowheads show double-labeled cells. Scale bar, 50 µm. d Percentage of V2Rp-positive VSNs among the ESP22-induced Egr1-expressing cells. e Representative immunohistochemical images of pS6-expressing VSNs in ESP22-stimulated V2Rp4^+/−, V2Rp4^−/−, or V2Rp5^−/− mice. Arrowheads represent example pS6-positive VSNs. Scale bar, 50 µm. f The number of pS6-positive cells per VNO section. For each genotype, 18 sections from each of 3 animals were quantified. ***p < 0.001, **p < 0.01, and *p < 0.05 by Steel-Dwass test. g–i Quantification of the rejection ratio using V2R mutant female mice pre-exposed to either control buffer or ESP22. The genotype of the female mice is shown at the bottom of each graph. Gray lines and dots indicate data from an individual animal. Error bars, S.E.M. ns, not significant. *p < 0.05 by Kruskal-Wallis test with Bonferroni correction in g or Wilcoxon signed rank test in h, i