Fig 1: Protein analysis of FGFR1FCPG and FGFR1FCSPG reveals FGF receptor interaction with focal adhesion complexes and intracellular trafficking. (A) GO term analysis of proteins identified from coimmunoprecipitation/mass spectroscopy. (Left panel) Top-level primary term distribution from 1418 proteins identified across all FGFR1 mutants and control. (Right panel) Fold enrichment of secondary and tertiary terms involved in endocytosis and ECM interactions. (B) Images of proximity ligation assay in E9.5 Fgfr1Myc/Myc, Fgfr1FCPG/FCPG, and Fgfr1FCSPG/FCSPG embryos. An anti-Myc antibody was paired with an antibody against the indicated protein to identify proximity of the two proteins within 40 nm. FAK, Src, PIK3R2, and EPS15 were chosen due to potential interactions with Fgfr1Myc/Myc at E8.5. Background fluorescence was filtered, and threshold values were standardized across all images. Nonspecific rabbit and mouse IgG primary antibodies were paired as a negative control. Scale bar, 100 μm. Black-and-white images depict 3× digital magnification of the boxed areas in the fluorescent images to show greater detail. (C) Quantification of PLA puncta in focal adhesion-related genes (left graph) and endocytosis-related genes (right graph). FAK, PIK3R2, and EPS15 all exhibited a significant increase in puncta compared with control samples in Fgr1Myc/Myc embryos, consistent with data from E8.5 (Supplemental Fig. S7A,B). Fgfr1FCPG/FCPG embryos exhibit a significant decrease in puncta formed with FAK compared with both wild type and Fgfr1FCSPG/FCSPG embryos. Both Fgfr1FCPG/FCPG and Fgfr1FCSPG/FCSPG embryos exhibit a significant reduction in puncta formed with PIK3R2 compared with wild type. Significance was determined using one-way ANOVA with Bonferroni correction for multiple comparisons. n = 3 biological replicates per genotype, with five sections imaged per embryo.