Fig. 2

Galectin-1 and -3 directly interact with sugar chains of FGFR1 extracellular domain. a, b Purified recombinant galectin-1 and galectin-3 were bound to NiNTA and incubated with cell lysates prepared from U2OS-R1 cells. Bound proteins were analyzed with western blotting. c U2OS-SBP-R1 cells were treated with FGF1 (50 ng/ml), heparin (10 U/ml) and subjected to fractionation into membrane, cytosolic and nuclear fractions. Subsequently co-purification of endogenously expressed galectin-1 with SBP-FGFR1 was determined with western blotting. d BLI analyses of FGFR1 interactions with galectins. The extracellular part of FGFR1c (FGFR1ecd-Fc) (10 μg/ml) was immobilized of Protein-A sensors and interaction with galectin-1 and galectin-3 (10–30 μg/ml) was monitored. The binding constants for galectin-1 and galecti-3 interaction with FGFR1ecd-Fc were determined with BLI. e Enzymatic de-glycosylation of FGFR1ecd-Fc with PNGase. FGFR1ecd-Fc (0.5 mg/ml) was incubated with PNGase F (0.3 U/ml) for 4 h at 37 °C. The enzymatic removal of sugar chains from FGFR1ecd-Fc was monitored with SDS-PAGE. f BLI analysis of galectin-1 and -3 interaction with PNGase treated FGFR1ecd-Fc. FGFR1ecd-Fc and PNGase treated FGFR1ecd-Fc (10–30 μg/ml) were immobilized on sample and reference Protein A sensors, respectively, and incubated with galectin-1 and galectin-3 (10–30 μg/ml). g BLI analysis of galectin-1 and galectin-3 interaction with the extracellular region of FGFR1 of bacterial origin (GST-FGFR1ecd). GST-FGFR1ecd (10 μg/ml) was chemically immobilized on AR2G sensors (both sample and reference) and incubated either with FGF2 (10 μg/ml) or galectin-1 and galectin-3 (10 μg/ml). h Competitive binding of galectin-1 and galectin-3 to FGFR1ecd-Fc. FGFR1ecd-Fc (10 μg/ml) was immobilized on Protein-A biosensors. Sample biosensors were incubated with galectin-3 (30 μg/ml) to saturate FGFR1ecd-Fc, whereas reference biosensor was incubated in the buffer only. Next, biosensors were incubated with galectin-1 (10 μg/ml) or galectin-1 (10 μg/ml)/galectin-3 (30 μg/ml) mixture (for sensors treated first with galectin-3 to avoid galectin-3 dissociation). FGFR1ecd-Fc (10 μg/ml) was immobilized on Protein-A biosensors. Sample biosensors were incubated with galectin-1 (30 μg/ml) to saturate FGFR1ecd-Fc, whereas reference biosensor was incubated in the buffer only. Next, biosensors were incubated with galectin-3 (10 μg/ml) or galectin-3 (10 μg/ml)/galectin-1 (30 μg/ml) mixture (for sensors treated first with galectin-1 to avoid galectin-1 dissociation). i Influence of galectins binding on the FGFR1 interaction with FGF2. FGFR1ecd-Fc (10 μg/ml) was immobilized on Protein-A biosensors. Next, sample biosensors were incubated with galectin-1 or galectin-3 (30 μg/ml) to saturate FGFR1ecd-Fc, whereas reference biosensor was incubated in the buffer only. Next, biosensors were incubated with FGF2 (10 μg/ml)