Fig. 6
Enhanced internalization of tetrameric FGF1-SA conjugates via FGFR1-mediated endocytosis. A Live cell imaging of the kinetics of FGF1E-AviTag-Biot and FGF1E-AviTag-Biot-SA-4A endocytosis. U2OS-R1 cells were incubated on ice for 40 min with Alexa Fluor 488 C5 maleimide-labeled FGF1E-AviTag-Biot in the presence or absence of SA-4A, shifted to 37 °C and imaged live for 60 min using spinning disk confocal microscopy. Images taken at the indicated time points are shown. Scale bar represents 50 μm. B Quantitative analysis of endocytosis of FGF1E-AviTag-Biot and FGF1E-AviTag-Biot-SA-4A. Average values from five independent live cell imaging experiments ± SEM are shown. t-test was used to assess statistical significance; *p < 0.05, **p < 0.01, ***p < 0.0001, n.s.- not significant. C Efficiency of FGF1E-AviTag-Biot and FGF1E-AviTag-Biot-SA-4A internalization analyzed by flow cytometry. Serum-starved U2OS-R1 cells were treated with Alexa Fluor 488 C5 maleimide FGF1E-AviTag-Biot and FGF1E-AviTag-Biot mixed with SA-4A. After 40 min incubation on ice, cells were transferred to 37 °C for 30 min, and then subsequently analyzed by NovoCyte 2060R Flow Cytometer. Average values of three independent experiments ± SD are shown. t-test was used to assess statistical significance (n = 3) * p < 0.05