Fig. 7. EHD1 tubular structures promote recycling of MHC-I to the cell surface. (A) Time-dependent co-localization of internalized MHC-I with EHD1 tubules by live image analysis. MHC-I monoclonal antibodies were coupled to Alexa Fluor 568 F(ab′)2 fragment of goat anti-mouse IgG. The coupled antibodies were then used to continuously pulse HeLa cells that were transfected 24 h earlier with a GFP–EHD1 construct. Images of MHC-I uptake (left panels) and GFP–EHD1 tubules (right panels) are depicted. Arrows (white) mark MHC-I tubular structures that appear at 15–20 min of internalization and co-localize with pre-existing GFP–EHD1 tubules (black arrows). Images are shown inverted to facilitate analysis (see Supplementary time-lapse video). Bar, 10 µm. (B) Quantification of EHD1-enhanced MHC-I recycling by a CELISA assay. HeLa cells were transfected with cDNA coding for H-2D^d (mouse MHC-I), H-2D^d and GFP–EHD1, H-2D^d and Myc-EHD1, H-2D^d and GFP–EHD1-G65R, or H-2D^d and GFP–EHD1-K220N. Internalization of MHC-I over time was monitored 24 h after transfection by CELISA utilizing a biotinylated anti-MHC-I antibody (see Materials and methods), and the fraction of MHC-I antibody on the surface at each time point was recorded. A representative experiment from four independent CELISA assays is depicted, with triplicates at each time point.