Fig. 1

Identification of FGFR1 partner proteins. a Cellular localization of SBP-FGFR1. U2OS-SBP-R1 cells were incubated with Streptavidin-AlexaFluor-555 and kept either at 4 °C or 37 °C. Cells were fixed and analyzed with fluorescence microscopy. Scale bars represent 50 μm. b Serum starved U2OS-R1 and U2OS-SBP-R1 cells were stimulated for 15 min with FGF1 (20–100 ng/ml) and heparin (10 U/ml). Cells were lysed and activation of cellular signaling was assessed with western blotting. c Purification of full-length SBP-FGFR1 from model mammalian cells. U2OS-R1 and U2OS-SBP-R1 cells were lysed under mild conditions and SBP-FGFR1 was recovered by affinity purification using streptavidin-agarose resin. Proteins were eluted with sample buffer and separated with SDS-PAGE and stained with CBB. d Western blotting analysis of SBP-FGFR1 affinity purification from U2OS-SBP-R1 cells. Denaturing elution was performed with SDS-PAGE sample buffer, while elution under native conditions was carried out with 4 mM biotin. e BN-PAGE analysis of SBP-FGFR1 purification. SBP-FGFR1 isolated from U2OS-SBP-R1 cells under non-denaturing conditions was subjected to 4–13% BN-PAGE. The migration of SBP-FGFR1 was analyzed with western blotting. f SBP-FGFR1 interacts with FGF1. U2OS-SBP-R1 and U2OS-R1 cells were incubated with FGF1, cells were washed, bound proteins were eluted with SDS-PAGE sample buffer and analyzed with western blotting. g Streptavidin-agarose pull down allows for purification of activated SBP-FGFR1. Serum starved U2OS-SBP-R1 cells were treated for 15 min with FGF1 (200 ng/ml) and heparin (20 U/ml). Next, cells were lysed, SBP-FGFR1 was isolated via affinity purification and activation of the receptor was assessed with western blotting. h. SBP-FGFR1 interaction network identified in affinity purification followed by mass spectrometry. Proteins were classified using STRING (http://www.string-db.org/). Blue spheres represent proteins involved in cellular protein transport, while red spheres depict proteins implicated in vesicle trafficking. i Verification of MS experiments. U2OS-SBP-R1 and U2OS-R1 cells were lysed and co-purification of selected proteins with SBP-FGFR1 was determined with western blotting