Fig. 3

Purification of functional SA-FGF1 oligomers. A Size exclusion chromatography of FGF1-AviTag-Biot-SA-4A oligomer. The absorbance spectra were monitored at 280 nm. B and C Analysis of purified FGF1-AviTag-Biot-SA-4A complex. To analyze the efficiency of the folding and purity of the complex, SDS-PAGE (B) and western blotting with antibodies recognizing FGF1 (C) were performed. To maintain the tetrameric form of the protein, prepared samples were not subjected to thermal denaturation. D and E Analysis of various purified FGF1-SA oligomers (sequentially from monomer to tetramer). The purified complexes were subjected to SDS-PAGE analysis (D) and western blotting with antibodies recognizing FGF1 (E). Both methods excluded thermal denaturation of samples to maintain the tetrameric form of proteins. F. FGF1-SA oligomers are able to activate FGFR1. Serum-starved NIH3T3 cells were incubated for 15 min with FGF1-AviTag-Biot-SA-1A3D (50 ng/mL), FGF1-AviTag-Biot-SA-2A2D (50 ng/mL), FGF1-AviTag-Biot-SA-3A1D (50 ng/mL) or FGF1-AviTag-Biot-SA-4A (50 ng/mL) and adequately higher concentrations of FGF1, as a control, to provide the cells with equal amounts of FGF1 targeting molecule. Proteins were added in the presence of heparin. Cells were lysed and activation of FGFR1, and receptor-downstream signaling (using antibodies recognizing phosphorylated key tyrosine within intracellular FGFR tyrosine kinase domain (pFGFR) and receptor-downstream ERK (detected with antibodies recognizing phosphorylated ERK (pERK) signaling was assessed with western blotting. The level of tubulin served as a loading control