Rac is not required for endocytosis of soluble EphB2-Fc. (A) Representative images showing the effects of siRNA depletion of Rac subfamily on EphB2-Fc endocytosis in ephrinB⁺ SKN cells. SKN cells were treated with indicated siRNAs before stimulation with fluorescently labeled, preclustered EphB2-Fc, fixed without permeabilization, and stained against Fc (surface EphB2, shown in red or yellow in the merge). Internalized vesicles appear as green puncta (total EphB2-Fc) within the vicinity of SKN nuclei (H2B channel, shown in blue). Bars: 10 µm; (inset) 2.5 µm. (B) Quantification of A, including Rac1 single depletion and hFc negative control. Images were analyzed with CellProfiler. Results are shown as mean ± SE (n = 4–6 independent experiments, >408 cells per condition per experiment, data normalized to median scramble value per experiment); ns, not significant; **, P < 0.01; ***, P < 0.001, one-way ANOVA with Dunnett’s post hoc test. (C) Quantification for effect of Rac inhibitor EHT1864 versus vehicle control on EphB2-Fc endocytosis in ephrinB⁺ SKN cells. Experimental design, analysis, and statistics are as described in A and B (n = 4–8 independent experiments, >345 cells per condition per experiment, data normalized to median vehicle value per experiment); statistics are as described in B. (D) Quantification of the effect of Rac inhibitor EHT1864 versus vehicle control on EphB2-Fc endocytosis in HeLa cells overexpressing ephrinB1-mCherry. Cells were stimulated with fluorescently labeled, preclustered EphB2-Fc fixed without permeabilization and immunostained against Fc (surface EphB2). Cells were imaged and manually scored for internal vesicles. Results shown as mean ± SE (n = 3–5 independent experiments, 29–128 cells per condition per experiment); statistics are as described in B. (E) Quantification of the effect of Rac inhibitor EHT1864 versus vehicle control on ephrinB2-Fc endocytosis in HeLa cells overexpressing EphB2-mCherry. Cells were stimulated with fluorescently labeled, preclustered ephrinB2-Fc or hFc, fixed, stained, and analyzed as in D (n = 3–5 independent experiments, 42–64 cells per condition per experiment, data normalized to median vehicle value per experiment); statistics are as described in B. (F and G) Representative images from D and E, respectively. Cell boundary outlined by blue border. Internalized vesicles appear as green puncta, distinct from surface signal (appears as yellow). Bars: 10 µm; (inset) 2.5 µm. (H and I) Representative images for Rac activation using RaichuEV Rac1 FRET biosensor either in wild-type (WT; H) or ephrinB2⁺ (I) HeLa cells after stimulation with EphB2-Fc. Cells were imaged before and 20 min after fluorescently labeled, preclustered EphB2-Fc stimulation. Top panels show pseudocolor intensity code. Bottom panel shows accumulation of EphB2-Fc; the cell boundary is outlined by a dashed red border. Bars, 10 µm. (J and K) Quantification of the relative difference in Rac activation between WT and test conditions (ephrinB2⁺ or EphB2⁺ cells) 20 min after stimulation with either EphB2-Fc (J) or ephrinB2-Fc (K), respectively. Results are shown as mean ± SE (n = 15–19 cells per condition, data relative to prestimulation time point per each condition); **, P < 0.01; Student’s t test. (L) Increase in Rac activity in EphB2⁺ and ephrinB1⁺ cells stimulated with ephrinB2-Fc or EphB2-Fc, respectively, relative to the extent of endocytosis (calculated from total fluorescent signal). Results are shown as mean ± SE (n = 15–19 cells per condition); ***, P < 0.001, Student’s t test. (M) Representative images for Rac activation using RaichuEV Rac1 Fret biosensor in EphB2⁺ HeLa cells after 20-min exposure to fluorescently labeled, preclustered ephrinB2-Fc. Fig. S3 B shows both prestimulation and the 20-min time point for comparison. Top panel shows pseudocolor intensity code. Bottom panel shows accumulation of ephrinB2-Fc; the cell boundary is outlined by a dashed red border. Bar, 10 µm.