Transferrin uptake and recycling

Transferrin uptake was measured by using pH-insensitive Alexa Fluor 488-transferrin as described previously (Kondapalli et al., 2015). Cells were starved in serum-free media for 30 min, followed by incubation with Alexa Fluor 488-transferrin (Thermo Fisher Scientific) (100 μg/mL) for 30 min at 37°C. For uptake kinetics, cells were incubated with Alexa Fluor 488-transferrin for 2, 5, 10, 15, and 30 min. For transferrin recycling kinetics, cells were incubated with Alexa Fluor 488-transferrin for 30 min, washed twice with ice-cold PBS pH 7.4 and incubated in normal media for 0, 5, 10, 15, and 30 min. Cells were then washed with ice-cold PBS pH 7.4 to remove excess transferrin, followed by ice-cold PBS pH 5.0 and pH 7.4 to remove surface bound transferrin. Transferrin uptake or recycling were quantified by analyzing ~10,000 cells using flow cytometry. Surface accessible transferrin receptors was determined by cell surface biotinylation. Starved cells were incubated with biotin conjugated transferrin (50 μg/mL, Thermo Fisher Scientific) for 30 min at 4°C and washed twice with ice-cold PBS pH 7.4 to remove excess transferrin. Cells were then lysed for western blot.