Early delivery of RUSH-α5 to the cell surface. (A) Representative immunofluorescence images of U2OS cells co-expressing RUSH-α5 (green) and the ER marker ERoxBFP (magenta) plated on FN (10 µg/ml) ± biotin treatment for the indicated times. Arrows indicate rapidly budding RUSH-α5–positive vesicles adjacent to cell protrusions (≤15 min after release) (see also Video 9). (B) TIRF imaging of RUSH-α5 after release (0 min). The polarized delivery to the cell surface at the protruding area can be observed from 15 min after release (see also Video 10). (C) Representative images of RUSH-α5 (green) and RUSH-CD59 (magenta) release in U2OS cells co-expressing both constructs and plated on dual-coated micropatterns (alternating FN coating (cyan) and collagen-peptide (GFOGER) (non-fluorescent) lines). Nuclei (blue) are co-labeled. White insets represent ROIs that are magnified for each channel. FN, fibronectin. (D) Flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in RUSH-α5–expressing U2OS cells ± biotin. Representative histograms and quantification from two independent experiments of cell surface GFP (ratio of the geometric means of the surface signal divided by the total GFP signal, normalized by subtracting the 0 min value) are shown.