Fig. 3: APEX2 tagged-FGFR2b and RAB11a identifies compartment-specific signalling partners upon FGF10 stimulation.
a Schematic underlying the spatially resolved phosphoproteomics (SRP) approach. b Immunoblot analysis (N ≥ 3 independent biological replicates) with the indicated antibodies of HeLa_FGFR2bST (right) or HeLa_FGFR2b-APEX2ST (left) stimulated with FGF10 for 1, 8, 40, 60 or 120 min. UT, treatment with vehicle as control. c Representative confocal images of FGFR2b (red) internalisation in the cytoplasm and FGFR2b recycling to the plasma membrane in HeLa_FGFR2b-APEX2ST, expressing eGFP-RAB11a (wtRAB11), dominant negative eGFP-RAB11a_S25N (DnRAB11) or dominant negative dynamin-2_K44A-eGFP (DnDNM2) (green), and treated with FGF10 for 40 min. UT, treatment with vehicle as control. Early endosome antigen 1 (EEA), blue. Scale bar, 5 μm. Zoomed images of the region indicated by the arrowheads (scale bar, 50 μm) and single channels for FGF10-stimulated cells for 0 and 40 min. are shown in the inset and on the right after the broken lines, respectively. White arrowheads indicate co-localisation and pink arrowheads indicate a lack of co-localisation. d Quantification of the co-localisation of stimulated FGFR2b (red pixels) with GFP-tagged proteins (green pixels) indicated by red-green pixel overlap fraction (top panel). Quantification of the co-localisation of FGFR2b (red pixels) with EEA1 (blue pixels) indicated by red-blue pixel overlap fraction (bottom panel). Representative images are shown in 3c. Values represent median ± SD from N = 3 independent biological replicates where we analysed between 2 and 5 cells for each N; ***p value <0.0005 (one-sided students t-test). e Immunoblot analysis (N ≥ 3 independent biological replicates) with the indicated antibodies of input or biotinylated proteins enriched with Streptavidin beads from HeLa_FGFR2b-APEXST treated with vehicle (UT), with H2O2, or with FGF10 for 1 and 8 min. f Schematic of FGFR2b localised at RAB11-positive recycling endosomes upon 40 min stimulation with FGF10. g Immunoblot analysis (N ≥ 3 independent biological replicates) with the indicated antibodies of input or biotinylated proteins enriched with Streptavidin beads from HeLa_FGFR2bST_RAB11-APEX2 stimulated with either H2O2 or with FGF10 for 40 min. UT, treatment with vehicle as control. Source data are provided as a Source Data file.
