Fig. 1

Glycosylation-deficient FGFR1 is localized to the nuclear envelope where it displays high level of autoactivation. A. Schematic representation of the structure of FGFR1 and FGFR1.GF. Positions of the N-glycosylation sites within the extracellular domain of the receptor are marked. B. FGFR1 activation in the presence or absence of FGF1 (100 ng/mL) in U2OS cells (control) and U2OS cells transfected with FGFR1 and FGFR1.GF. Tubulin served as a loading control. Quantification of pFGFR/FGFR1 signals was performed with ImageLab 5.0 software. Average values of 3 independent experiments (n = 3) ± SD are shown. Statistical analyses were performed with Student’s t-test (*p < 0.05; **p < 0.005 and ***p < 0.001; n.s. – not significant). C. Internalization of fluorescently labeled FGF1 into U2OS, U2OS-R1 and U2OS-R1.GF cells. Cells were incubated with FGF1-DL550 (500 ng/mL) and heparin (10 U/uL) for 30 min at 37 °C, cells were fixed, nuclei were stained with NucBlue dye and cells were analyzed by fluorescence microscopy (n = 3). Scale bar represents 10 µm. D. Pull down experiment demonstrating interaction between FGF2 and SBP-FGFR1 or SBP-FGFR1.GF. U2OS cells stably producing receptor variants were lysed, receptor was captured on streptavidin-agarose beads and beads were then incubated with recombinant FGF2. After extensive washing, co-purification of FGF2 with SBP-FGFR1 and SBP-FGFR1.GF was analyzed by western blotting. Empty beads served as a control for specificity of FGF2 purification with FGFR1 variants (n = 3). E. Subcellular localization of FGFR1 and FGFR1.GF. FGFR1 variants in transiently transfected U2OS cells were detected by immunofluorescence in permeabilized and non-permeabilized cells. Nuclei were labelled with NucBlue dye (n = 3). Scale bar represents 10 µm. F. Immunofluorescence-based co-localization of SBP-FGFR1.GF with the ER/Golgi marker protein COPB (n = 3). Scale bar represents 20 µm. G. Immunofluorescence-based co-localization of SBP-FGFR1.GF with pFGFR signal (n = 3). Scale bar represents 20 µm. H. Immunofluorescence-based co-localization of SBP-FGFR1.GF with the nuclear envelope marker proteins lamins A/C (n = 3). Scale bar represents 20 µm. I. PLA-based analysis of SBP-FGFR1.GF interaction with lamins A/C in the nuclear envelope (n = 3). Scale bar represents 20 µm. J. Localization of FGFR1 in U2OS-R1 cells (control) and in U2OS-R1 cells treated with tunicamycin (n = 3). Scale bar represents 20 µm