RUSH-α5 delivery and localization following release. (A) RUSH-α5-pHluorin released in U2OS co-expressing Paxillin-mScarlet on FN- and anti-GFP antibody-coated surfaces. The intensity of RUSH-α5-pHluorin signal was quantified in and outside adhesions (paxillin positive, represented in the insets). Data are mean ± SD, N = 12 cells (N = 6 cells from 1 experiment for T = 45 min), pooled from 2 independent experiments. Ordinary one-way Anova with Holm-Šídák’s multiple comparisons test; data distribution was assumed to be normal but this was not formally tested. Scale bars: 10 µm (main and insets). (B) High resolution imaging of RUSH-α5 after 15 min of release in U2OS. PDI (ER marker) or GM130 (Golgi marker) are co-stained. Arrows in the insets indicate RUSH-α5 positive vesicles. Scale bar: 10 µm (main), 5 µm (insets). (C and D) High-mannose integrin-α5 is delivered to the cell surface. (C) Flow cytometry analysis of high-mannose proteins at the cell surface detected with the fluorescent lectin PFL647 in U2OS cells expressing RUSH-α5, without release and 1 h after release. The left panel shows histograms of one experiment, the right panel shows the geometric fluorescence mean of the PFL647 signal for individual experiments (N = 2 independent experiments). (D) U2OS expressing RUSH-α5 were labeled at their surface after 1 h release with a lectibody specifically recognizing high-mannose proteins. The lectibody was then pulled down by protein G beads. This Western blot shows GFP detection in the pull-down, indicating the presence of high-mannose RUSH-α5 at the cell surface after release. Representative of N = 3 independent experiments. Source data are available for this figure: SourceData FS3.