Fig 1: FAM3B binds to FGFR and activates ERK through FGFR. (A) FAM3B binds to full-length FGFR1 in Xenopus embryos. Two-cell-stage embryos were injected with FAM3B-Flag mRNA (400 pg) and wild type (WT) or kinase-inactive mutant (KI) FGFR1-HA mRNA (400 pg) separately into different blastomeres. Gastrula-stage embryos were harvested for immunoprecipitation with Flag beads followed by immunoblotting with Flag and HA antibodies. Total protein expression was confirmed by immunoblotting of the input. (B) FAM3B binds to the ectodomain of FGFR1 in solution. CM for FAM3B-HA and ectodomain of FGFR1 (FGFR1-Ecto-Flag) were combined and allowed to bind, followed by immunoprecipitation with Flag beads and subsequent immunoblotting with Flag and HA antibodies. Protein expression in CM was confirmed by immunoblotting of the input. (C) FAM3B does not bind to ß-Klotho ectodomain. CM for the ectodomain of ß-Klotho (ß-Klotho-Ecto-HA) and FAM3B-Flag were combined and allowed to bind. Total protein expression in the CM was confirmed by immunoblotting of the input. This experiment serves as a negative control for binding to the extracellular domain of FGFRs. (D–G) FAM3B binds to FGFR ectodomain in vitro. Purified rhFAM3B protein (1 µg) was incubated with Fc-tagged human FGFR ectodomain protein (1 µg), including FGFR1-Ecto-Fc (D), FGFR2-Ecto-Fc (E), FGFR3-Ecto-Fc (F), and FGFR4-Ecto-Fc (G) as indicated. Then the protein mixture was subjected to pull-down with Protein A magnetic beads and subsequent immunoblotting with Fc and His antibodies. Protein expression was confirmed by immunoblotting of the input. PD: pull-down; PA: Protein A magnetic beads. (H) FAM3B CM rapidly activates ERK. HEK293T cells were serum starved for 24 h and treated with serum-free control or FAM3B CM for the indicated times. Cells were harvested for immunoblotting with pERK and total ERK antibodies. a-Tubulin served as a loading control. (I) FAM3B CM-induced ERK activation is blocked by FGFR inhibition. HEK293T cells transfected with FGFR1 KI or not were serum starved for 24 h and pretreated with FGFR inhibitors SU5402 (20 µM), AZD4547 (1 µM), Erdafitinib (1 µM), or Ly2874455 (1 µM) for 2 h. Then cells were stimulated with serum-free control CM or FAM3B CM in the presence of these inhibitors for 20 min and harvested for immunoblotting with the indicated antibodies. a-Tubulin served as a loading control. (J) FAM3B CM activated an FGF reporter derived from the mouse Dusp6 promoter (40), which was blocked by FGFR1 inhibition. HEK293T cells were transfected with FGF Luciferase reporter together with Renilla and FGFR1 KI as indicated. Twenty hours after transfection, cells were serum starved for 20 h, followed by treatment with serum-free control or FAM3B CM for 8 h. Experiments were performed in triplicate. Data are mean ± SD. Statistical significance was assessed by unpaired two-tailed Student’s t test. ***P < 0.001. RLA, Relative Luciferase Activity.