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Fig. 3 | Cell Communication and Signaling

Fig. 3

From: Differential regulation of fibroblast growth factor receptor 1 trafficking and function by extracellular galectins

Fig. 3

Extracellular galectins regulate spatial distribution of FGFR1. a Impact of galectin-1 and -3 on the cellular trafficking of SBP-FGFR1. Serum starved U2OS-SBP-R1 cells were pre-incubated with Streptavidin-AlexaFluor-555 (4 μg/ml) for 5 min on ice to label SBP-FGFR1. Next, galectins were added to the cells (10 μg/ml) and cells were in some cases treated with FGF2 (200 ng/ml) and heparin (20 U/ml) for 1 h at 37 °C. Cells were subsequently fixed, nuclei were labeled and cells were analyzed with fluorescence microscopy. Scale bars represent 50 μm. b Co-localization of galectins with FGFR1. For analysis of galectin-1-DyLight488 and galectin-3-DyLight488 co-localization with SBP-FGFR1, U2OS-SBP-R1 cells were pre-incubated with Streptavidin-AlexaFluor-555 (4 μg/ml) for 5 min on ice to label SBP-FGFR1. Next, fluorescently labeled galectins were added to the cells (10 μg/ml) for 1 h at 37 °C and analyzed with fluorescence microscopy. Arrows mark selected points of co-localization. c Influence of galectins on the trafficking of FGFR1. For analysis of the impact of galectins on FGF2 internalization, serum starved U2OS-R1 cells were incubated with galectins (10 μg/ml) for 10 min at RT and subsequently with FGF2-DyLight-550 (2 μg/ml) and heparin (20 U/ml). d BN-PAGE analysis of the impact of galectins on the clustering of FGFR1ecd-Fc.Purified extracellular region of FGFR1 (FGFR1ecd-Fc (2 μg)) was incubated with recombinant galectin-1 or galectin-3 (2–5 μg) in PBS for 15 min at RT and proteins were separated on 4–13% gradient BN-PAGE gels. Proteins were transferred onto PVDF membrane and detected with anti-Fc antibodies, stripped and detected with anti-galectin-3 and anti-galectin-1 antibodies

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