Anti fgf2

The binding of MPS IIIA GAGs to FGF2 was analysed in the Blitz ® system (ForteBio) using protein G sensors and operated with PBS, pH 7.4 containing 1% (v/v) Tween-20. Sensors were equilibrated in buffer for 30 s and then exposed to anti-FGF2 antibody (1:1000, rabbit polyclonal anti-FGF2, R&D Systems) for 120 s followed by rinsing in buffer for 30 s. Sensors were exposed to 50 μg/mL FGF2 for 120 s followed by rinsing in buffer for 30 s and then exposed to MPS IIIA GAGs or heparin (1–20 μg/mL; Sigma H3393) for 120 s and rinsed with buffer for 30 s. The affinity, K_D, between FGF2 and either MPS IIIA GAGs or heparin was determined assuming 1:1 binding. Signalling via the fibroblast growth factor receptor (FGFR) was determined using the BaF32 cell proliferation assay which relies on the formation of an active ternary complex between GAG, FGF2 and FGFR1c [44] (link). The assay was performed as described previously [45] (link) in the presence of 0.03 nM FGF2 and either unfractionated heparin (0–0.5 μg/mL; Sigma H3393) or MPS IIIA GAG (0–2 μg/mL). The relative number of cells present was determined using the MTS reagent (Promega, Madison, Wisconsin, USA) by addition to the cell cultures for 6 h prior to measurement of absorbance at 490 nm.