a, Transwell migration of lipopolysaccharide-activated GPR174-sufficient or -deficient B cells in response to LysoPS of indicated concentrations. Data are plotted as mean ± s.e.m. of the percentage of cells that migrated in triplicated wells at each concentration, fitted with three-parameter log dose–response curves; two-way ANOVA was used to compare the two groups. One of two independent experiments with similar results is shown. b–d, Transwell migration of lipopolysaccharide-activated B cells that were transduced with a control or GPR174-expressing vector, in response to stroma-conditioned medium (stroma), dimethylsulfoxide (DMSO) control or LysoPS of indicated concentrations (b); or in response to stroma-conditioned medium (stroma), conditioned medium heated at 100 °C for 10 min (heated), conditioned medium treated with the proteinase K inhibitor PMSF alone (PMSF), conditioned medium treated with proteinase K and then PMSF (PK + PMSF), or conditioned media treated as above and then further supplemented with 300 ng ml−1 CXCL13 (PK + PMSF + CXCL13) to exclude cell damage owing to remaining proteinase activity (c); or in response to blank culture medium, or mouse or porcine stroma-conditioned media (d). All data are plotted as the mean percentages of cells that migrated in triplicated wells, from one experiment representative of three with similar results. Two-way ANOVA with Bonferroni’s multiple comparison tests were used to compare vector and GPR174 groups, with P values given in the graphs. ****P < 0.0001; ns, not significant. e, Workflow showing the six-step biochemical fractionation method for identifying GPR174 ligands in splenic stroma-conditioned medium. See Methods for details. CV, column volume. f–k, Chromatography traces and putative ligand activities as detected by the transwell assay in d in relevant fractions during the six steps of e. AU, arbitrary units; mS, millisiemens. l, Left, silver staining of the indicated fractions from k resolved on 12% SDS–PAGE; the black box on fraction 5 marks bands subjected to liquid chromatography-mass spectrometry (LC-MS)/MS analysis. Right, identified unique porcine CCL21-derived peptides (solid underlines) aligned against porcine, human and murine CCL21 protein sequences. For gel source data, see Supplementary Fig. 1. Data shown in f–l are from one of two independent experiments with similar results. FT, flow-through. Source Data