Alexa fluor 647 labeled egf

A549 cells were serum-starved for 3 h by incubation with starvation medium. Then, cells were incubated for 30 min with small molecules (dynasore: 100 μM, regorafenib/sorafenib: 3 μM) or an equivalent of DMSO, respectively, under normal culture conditions. Afterwards, cells were transferred to 4°C and washed twice with cold PBS and incubated with 100 ng/ml Alexa Fluor 647-labeled EGF (Thermo Fischer) and 25 μg/ml Alexa Fluor 488-labeled transferrin (Thermo Fischer) diluted in starvation medium for 1 h at 4°C. Cells were pulsed for 10 min at 37°C, washed three times with PBS and chased for different periods of time (0, 30, 60, and 120 min) in starvation medium containing small molecules but lacking EGF and transferrin. After the chase period, cells were transferred to 4°C and washed once with cold acid buffer (150 mM NaCl, 25 mM NaOH, pH 3.5) to remove surface-bound EGF and transferrin. Then, cells were washed twice with cold PBS, harvested by scraping and precipitated by centrifugation (1200 x g, 5 min, 4°C). Finally, cells were fixed with 4% paraformaldehyde and analyzed for Alexa Fluor 647 and Alexa Fluor 488 intensities as described for ‘Analysis of virus attachment’. Three independent experiments were performed in duplicates and per duplicate measurement, 10,000 cells were analyzed by flow cytometry.