Figure 4.

FPR1 is a functional receptor of FAM19A4. (a) FAM19A4 was tested for its chemotactic abilities on the HEK293-FPR1 cells, and significant differences compared with the medium were calculated. (b) In the cross-desensitization chemotaxis assay, fMLF and FAM19A4 were tested for their ability to chemoattract HEK293-FPR1 cells with or without pre-treatment with fMLF or FAM19A4 for 1 h and the chemotaxis index was calculated. (c) fMLF (0.1 µM) or FAM19A4 (0.1 µM) were used to treat the HEK293 cells expressing FPR1-EGFP for 1 h; these cells were then analyzed by confocal microscopy. The scale bars represent 25 µm. (d) After HEK293-FPR1 was stimulated with fMLF, FAM19A4 or the negative controls in suspension for 1 h, the quantity of FPR1 on the cell surface was evaluated using flow cytometry. The internalization rate of FPR1 and significant differences compared to the control were calculated. (e) In the saturation experiment, equivalent quantities of ¹²⁵I-FAM19A4 were incubated with varying quantities of the FPR1-transfected cell membrane extract in binding buffer. The y-axis represents the radioactivity of the binding, and the x-axis represents the concentration of FPR1. (f) In the competitive binding assays, equivalent quantities of the cell membrane extract and ¹²⁵I-FAM19A4 were incubated with varying quantities of unlabeled ligands. The y-axis represents the percentage of the max radioactivity of the specific binding complexes, and the x-axis represents the logarithmic form of the concentration of the competitors. (h, i) Boc-MLF (1 µM) was used to pre-treat the human (g) or mice (h) macrophages for 10 min followed by incubation with FAM19A4 for 30 min; the phagocytosis assay (g) or the ROS detection (h) were then performed as described before. fMLF, formyl-methionyl-leucyl phenylalanine; FPR1, formyl peptide receptor 1.