Fig. 1: Mouse VSNs activated by C57BL/6 male mice blood hemoglobin.

a Representative immunohistochemical image (left) and number of c-Fos-positive cells per VNO section from male mice stimulated by blood (1 µl) or control buffer (right). n = 3 for control and blood. Error bars, S.E.M. Arrowheads represent example c-Fos-positive cells. Scale bar, 100 µm. b Representative immunohistochemical images of anti-pS6 and anti-Gαo staining (left) and number of total pS6-positive cells in the Gαo and Gα_i2 zone of the VNO section from blood- or control buffer- stimulated male mice (right). Arrowheads represent example pS6-positive cells (left) and double-positive VSNs (right). n = 3 for control and blood. Error bars, S.E.M. Scale bar, 100 µm. c Representative immunohistochemical image (left) and number of total c-Fos-positive cells in the glomerular layer (Gl) and mitral/tufted cell layer (M/T) of the AOB section from male mice stimulated by blood (1 µl) (right). Arrowheads represent c-Fos positive cells. n = 3 for control and blood. Error bars, S.E.M. Arrowheads represent example c-Fos-positive cells. Scale bar, 100 µm. d The number of c-Fos-expressing cells in the M/T cell layer of the AOB sections from male mice stimulated with the indicated amount of blood. n = 3 for 0, 0.01, 0.03, 0.3, and 3 µl, and n = 4 for 0.1, 1, and 10 µl. Error bars, S.E.M. e Separation of blood by centrifuge. Separated blood components: plasma, cell lysate and residue were used for SDS-PAGE analysis and a c-Fos-inducing assay in C57BL/6 male mice. n = 3. Error bars, S.E.M. f, g Two-step HPLC purification with DEAE (f) and C4 columns (g). Chromatogram and c-Fos-inducing activity of each fraction are shown. The cell lysate fraction was used for DEAE column chromatography and the resultant active fraction was used for C4 column chromatography. The three fraction peaks were defined as heme, α-globin and β-globin by absorption spectrometry and mass spectrometry (Supplementary Fig. 1b-d). h c-Fos-inducing activity of recombinant β-globin. n = 3. Error bars, S.E.M. Scale bar, 100 µm. Arrowheads highlight example c-Fos-positive cells in the VNO (top) and in the M/T cell layer of the AOB (bottom). i Representative EVG recording of hemoglobin-dependent negative change in local field potential of the VNO. j Dose-dependent electrical responses of male VNO to hemoglobin. n = 10. Error bars, S.E.M.