Extended Data Fig. 6: Gremlin1 binds to FGFR1 in a way different from FGF1/FGFR1 interaction.
(a) The specific binding of Gremlin1 to immobilized FGFR1 is competed by soluble FGFR1 in a dose-dependent manner (n = 2 technical replicates, experiments have been repeated twice). (b) Soluble FGFR1 (200 ng/ml) competitively inhibits the activation of FGFR1/MEK/ERK signaling by Gremlin1 (100 ng/ml) in PC3. The experiments were repeated at least 3 times with similar results. (c) Schematics of FGFR1 mutations. (d) FGFR1-C176G or FGFR1-R248Q mutation abolishes co-immunoprecipitation of FGF1 and FGFR1(left panel), but do not influence the forming of protein complex between Gremlin1 and FGFR1(right panel). The experiments were repeated at least 3 times with similar results. (e-h) The binding between Gremlin1 and FGFR1 is not affected by addition of FGF1, and vice versa, which are revealed by Fortebio (e), co-immunostaining (f), pull-down (g), and immunoblotting (h) assays. Scale bars = 10 μm. The Fortebio experiment was repeated at least twice with similar results. The pull-down and immunoblotting assay were repeated at least 3 times with similar results. The immunostaining experiment was repeated at least twice. Multiple fields of cell culture slides were examined during each repeat and representative images were shown. (i) mRNA levels of GREM1 and FGFs in CRPCs (data were obtained from the Beltran 2016 dataset (dbGaP, phs000909), n = 49 tumor samples). (j) Correlation analysis of GREM1 and FGFs expression in CRPCs (data were obtained from the Beltran 2016 dataset (dbGaP, phs000909), n = 49 tumor samples). (k) The concentration of Gremlin1 and FGF1 in prostatic fluid of human PCa patients is analyzed by the enzyme-linked immunosorbent assay. n = 18 prostatic fluid samples in Gremlin1 ELISA assay; n = 23 prostatic fluid samples in FGF1 ELISA assay. (Two-tailed Student’ s t test was used for the statistical analysis. Data are presented as means ± s.e.m.).
