Gulp1 interacts with EphB2. (A) Validation of the interaction between Gulp1 and EphB2 using coimmunoprecipitation. HeLa cells overexpressing GFP-Gulp1 and either full-length Flag-EphB2 (Flag-EphB2-FL) or Flag-EphB2ΔC were either not treated (control) or treated with either preclustered Fc or preclustered ephrinB1-Fc, or cocultured with HeLa cells overexpressing ephrinB1ΔC-CFP (ephrinB1ΔC). (B) Model of EphB/ephrinB forward trogocytosis. EphrinBΔC from donor cells is trans-endocytosed into EphB⁺ responder cells. (C) Representative images showing forward trogocytosis in U251 cells (magenta dashed line, labeled with Cell-tracker) cocultured with ephrinB1ΔC-GFP⁺ donor HeLa cells (right panels, green dashed line), but not control GFP⁺ cells (left panels, green dashed line). Internalized ephrinB1ΔC vesicles in U251 cells were detected as green puncta void of surface HA-antibody labeling (arrows). Scale bars, 10 µm. (D) Validation of the interaction between endogenous Gulp1 and EphB2. U251 cells were first cocultured with either control GFP⁺ or ephrinB1ΔC-GFP⁺ HeLa cells for 30 min, and cell lysates were then subjected to immunoprecipitation by anti-EphB2 antibodies. (E) Representative images from live imaging of forward trogocytosis in HeLa cells. EphrinB1ΔC-mCherry⁺ donor cells were cocultured with responder cells expressing untagged EphB2 and GFP-Gulp1. Middle row: green dashed lines indicate the responder cell outline. Bottom rows show time course at time of contact and scission. Arrows indicate ephrinB1ΔC cluster formation and subsequent vesicle internalization. GFP-Gulp1 images pseudo-colored as heat maps (bottom row). Scale bars, 10 µm (top panel), 5 µm (inset), and 2 µm (time-lapse images). Elapsed time shown as min:s. (F) Average fluorescent intensities at contact sites of cluster formation (F′) and subsequent vesicles (F″) from time-lapse imaging of cocultures as described in E. Data displayed as heat maps of average intensity calculated over every event, with donor contact sites for each set aligned to top and center, and all images normalized to their respective background signal. Graphs show mean ± SEM of relative fluorescent units (RFU) changes through the central four pixels for the x axis. Data acquired from 32 events from 10 cells over two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; one-way ANOVA with Bonferroni’s multiple comparisons test performed on GFP-Gulp1 signal.