Alexa 488 conjugated transferrin

HEK293 cells stably expressing hZIP4-HA were grown in 24-well trays for 16 h on sterile glass coverslips. For the basal condition, the cells were incubated in the basal medium with 4 μg/ml anti-HA antibodies at 37°C for 30 min. For the other conditions, the cells were first treated with 20 μM TPEN for 10 min at 37°C, washed one time with the Chelex-treated medium and then incubated for 30 min at 37°C in the Chelex-treated medium containing 4 μg/ml anti-HA antibodies without or with 10 μM ZnCl₂. For transferrin internalization assay, the cells were treated in the same way under the same condition, except that 25 μg/ml Alexa 488 conjugated transferrin (Thermo Fisher Scientific,Cat# T13342) was added in the medium instead of anti-HA antibodies. The cells were washed twice with 1 mL of ice-cold DPBS and fixed for 10 min at room temperature using 4% formaldehyde. They were then permeabilized and blocked for 1h with DPBS containing 5% goat serum (Cell Signaling Technology, Cat# 5425S) and 0.1% Triton X-100 and then incubated with Alexa-568 goat anti-mouse antibodies at 1:500 (Thermo Fisher Scientific, Cat# A-11004, RRID:AB_2534072) at 4°C for overnight. After three washes with DPBS, coverslips were mounted on slides with fluoroshield mounting medium with DAPI (Abcam, Cat# ab104139). Samples were viewed with a 63X objective using a Zeiss Axio fluorescence microscope.