Transferrin alexa 647

For uptake of transferrin ligand, cultured rat neurons were starved for 10 min in HBS at 37 °C, and then incubated with transferrin–Alexa647 (Thermo Fischer Scientific, T23366,) at 50 μg ml⁻¹ in HBS for 10 min at 37 °C. After a quick wash with cold HBS, cells were stripped of surface-bound transferrin–Alexa647 with glycine buffer (100 mM NaCl, 50 mM glycine, pH 3, 300 mOsm) twice, rinsed with cold HBS and fixed with 4% paraformaldehyde/4% sucrose in PBS at room temperature for 15 min. For surface labelling, neurons were incubated with transferrin–Alexa647 for 10 min at 4 °C, followed by a quick wash with cold HBS and fixed. The cells were then permeabilized with PGT and labelled with anti-TfR antibody (1:1,000) (clone H68.4, Thermo Fisher Scientific, 13-6800, RRID AB_2533029) followed by anti-mouse Alexa568 conjugate (1:500) (Thermo Fisher Scientific, A11004, RRID AB_2534072). The samples were mounted in Fluoromount-G with DAPI and were imaged on a spinning-disk confocal microscope with a 63× objective and 488, 561 and 634 nm illumination. A stack of focal planes, 0.5 μm apart, was acquired for the GFP, Alexa568 and Alexa647 channels. A maximum-intensity projection of all channels was used for quantification of fluorescence measurements. We defined a mask of the cell in the Alexa568 channel and used it for quantification of transferrin–Alexa647 labelling.

RPE cells expressing epsin mutants were serum-starved for 4 h. Cells were subjected to hypo- and iso-osmotic shock for 10 min and then allowed to uptake transferrin Alexa 647 (25 µg/ml) (Thermo Fisher) for a further 10 min, followed by acid wash (acetic acid buffer pH 3.0) and immediate fixation with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min. For imaging surface receptor-ligand co-localization, acid wash was not performed. The intensity of transferrin Alexa Fluor 647 was calculated using epifluorescence signal and normalized to bulk mCherry-clathrin intensity.