Fig. 6
Rate of endocytosis of NHE6 ΔES is reduced in AP-1 cells. a AP-1 cells were transiently transfected with 3FNHE6v1HA WT (upper panels) or ΔES (lower panels) for 36 h, serum-depleted and then incubated with Alexa Fluor488-conjugated transferrin (Tf-AF488) for 45 min. Cells were then placed on ice and incubated with primary mouse monoclonal anti-Flag antibody and Alexa Fluor568 conjugated secondary antibody, and either fixed in 2 % paraformaldehyde immediately (0 min time point) or after incubation at 37 °C for 60 min. Footprints of the cells within the field of view are indicated as white dotted outlines and were derived from the transmitted light images (far left panels). After 60 min of internalization, the signals for the WT transporter highly colocalized with those for Tf-AF488, whereas the ΔES mutant showed only partial overlap (the white arrows heads indicate some overlapping punctate signals). Scale bars represent 10 μm. b Kinetics of endocytosis measured in AP-1 cells transiently expressing 3FNHE6v1HA WT or ΔES using a cell-based enzyme-linked immunosorbent assay (ELISA). Thirty-six hours post-transfection, surface NHE6 was labeled on ice using a mouse monoclonal anti-Flag antibody, followed by internalization of the Flag-labeled NHE6 at 37 °C for the indicated time points. Remaining cell surface NHE6 was labeled with a goat anti-mouse HRP-conjugated secondary antibody and detected using the fluorescence Amplex®Red substrate. Data points represent mean ± S.E.M. of four different experiments, each done in triplicate. Statistical significance (p < 0.05) was evaluated using a paired two-tailed Student’s t-test and indicated by an asterisk