Fig. 7: Phosphorylated ULK1 is recruited at the recycling endosomes.
a, b, f, g, k Immunoblot analysis with the indicated antibodies of HeLa_FGFR2bST_RAB11-APEX2 (a), T47D transfected with RAB11-APEX2 (T47D_RAB11-APEX2) (b), HeLa_FGFR2bST (f) or T47D (g) transfected either with wtRAB11, DnRAB11 or DnDNM2 (f, g), T47D transfected with GFP or DnRAB11 (k) stimulated with FGF10 for the indicated time points. Non proximal and proximal samples represent the supernatant and the pulldown following enrichment of biotinylated samples with streptavidin beads, respectively, and run against total lysates (total) (a, b). N ≥ 3 independent biological replicates. UT, treatment with vehicle as control. c Quantification of proximity ligation assay (PLA) puncta between FGFR2b and S638 pULK1 in HeLa_FGFR2bST cells treated with vehicle (UT) or FGF10 for 40 min.; p value <0.0005 *** (one-sided Students t-test). N = 9 independent biological replicates. d Representative confocal images of co-localisation between FGFR2b-APEX2 (red) and phosphorylated ULK1 on S638 (blue) in T47D_FGFR2KO_FGFR2b-APEXST transfected with RAB11 or GFP-DnRAB11 (green) and stimulated with FGF10 for 40 min. Scale bar, 5 µm. Inset, zoomed images of the region indicated by the arrowheads (scale bar, 50 μm). The white arrowhead indicates co-localisation, and the pink arrowheads indicate a lack of co-localisation. e, h, j, i Quantification of the presence (pixel proportion) and of the co-localisation (pixel overlap fraction) of the indicated proteins. 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 Student t-test). Representative images are shown in Fig. 7d (e), Supplementary Fig. 7f (h), Supplementary Fig. 7g (i) and Supplementary Fig. 7h (j). Source data are provided as a Source Data file.
