Fig. 2: Phosphoproteomics analysis identifies FGFR2b internalisation- and recycling-dependent signalling pathways.

a Workflow of the phosphoproteomics experiment in HeLa cells transiently expressing FGFR2b (HeLa_FGFR2b) and either GFP, DnRAB11 or DnDNM2 and treated with FGF10 for 40 min. UT, treatment with vehicle as control. b Principal component analysis (PCA) of the HeLa phosphoproteome from 2a showed small variation between technical replicates and separated samples based on experimental conditions. c t-distributed stochastic neighbour embedding (t-sne) analysis identified 11 clusters corresponding to phosphorylated peptides differentially regulated among four conditions. Colour corresponds to the cluster with the highest membership score, determined using fuzzy c-means clustering based on the median z-score of the four conditions. Each cluster is identified by a unique colour and corresponding number. Opacity corresponds to the membership score assigned to each phosphorylated site within its most likely cluster. Membership scores and t-sne coordinates are available in Source Data File. d Plots of the median z-scored intensities of phosphorylated sites based on the 11 clusters from Fig. 2c identified membrane response (red; clusters 3 and 4), internalisation response (light blue; clusters 5 and 8), and recycling response (dark blue; clusters 9 and 11). Colour key indicates membership value assigned by Fuzzy c-means clustering. e Plots of the median z-scored intensities of phosphorylated sites based on the three main clusters identified in Fig. 2d. HeLa_FGFR2b (yellow) and T47D (dark green) were treated as in Fig. 2a and Supplementary Fig. 2h, respectively. f KEGG pathway enrichment (calculated with Fisher’s exact test and FDR adjustment) between HeLa_FGFR2b and T47D phosphorylated proteins within the membrane (red), internalisation (light blue) and recycling (dark blue) responses (Fig. 2d) identified mTOR signalling as associated with FGFR2b recycling. The size of dot indicates statistical significance based on p value. g Visualisation of the subcellular localisation of the phosphorylated proteins belonging to the mTOR signalling pathway KEGG term in HeLa_FGFR2b (yellow) and T47D (green) using SubCellularVis³⁸.