Figure 8.
Figure 8. C-terminal modules of EphB2 have a negative regulatory effect on ephrinB2-induced clustering. (A) Steady-state fluorescence anisotropy of ephrinB2-induced EphB2 clusters. COS-7 cells transiently expressing wtEphB2-mGFP or EphB2ΔSAM/PBM-mGFP were treated with 0.5 µg/ml unclustered ephrinB2-Fc. Anisotropy values of representative cells before (left) and 20 min after (right) stimulation with ephrinB2 are shown. Deletion of SAM and PBM domains leads to a decrease of fluorescence anisotropy (i.e., increase in clustering). Bars, 10 µM. The color coding of anisotropy values is shown on the right. (B) Quantification of steady-state fluorescence anisotropy plots from before and after stimulation (20 min) with 0.5 µg/ml of unclustered ephrinB2-Fc. Data represent mean anisotropy ± SEM of n = 35 and 46 cells for wtEphB2-mGFP and EphB2ΔSAM/PBM-mGFP, respectively. Post-stimulation curves are significantly different from each other; ***, P < 0.001; Mann-Whitney nonparametric test. (C, top) Representative Western blots of anti-Flag–immunoprecipitated wtEphB2 or EphB2ΔSAM/PBM (as indicated) after stimulation with equal concentration of Fc control or ephrinB2-Fc (0.5 µg/ml; t = 20 min) using mouse anti-phosphotyrosine antibody; blots were stripped and reblotted for total EphB2 protein levels. (bottom) Quantification of autophosphorylation (**, P < 0.01; ***, P < 0.001; unpaired t test; n = 7 separate experiments). (D) Quantification of collapse responses of HeLa cells expressing either wtEphB2 or EphB2ΔSAM/PBM induced by equal concentrations of unclustered human-Fc or ephrinB2-Fc (1 µg/ml is shown; similar results were obtained with 0.5 µg/ml). Cell collapse was scored by measuring cell surface area. Data are shown as mean cell area ± SEM from n = 25 cells per condition. Statistical significance was determined as in Fig. 4 A. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (wtEphB2 versus EphB2ΔSAM/PBM; n = 8 separate experiments). (E) Quantification of EphB2 surface proteins by immunostaining of individual wtEphB2 (n = 15 cells) and EphB2ΔSAM/PBM (n = 15 cells) transfected cells. Cells were fixed and stained with mouse anti-Flag antibody to visualize EphB2 expressed at cell membranes. Total EphB2 protein levels were visualized by YFP fluorescence. Ratios of surface/total fluorescence intensity integrated over the whole cell body were measured as an indication for expression levels at the cell surface. Scatter in range of EphB2 expression at the cell surface was similar for both conditions with a slight trend toward lower EphB2 level for EphB2ΔSAM/PBM-transfected cells. The experiment was repeated three times with the same outcome.

C-terminal modules of EphB2 have a negative regulatory effect on ephrinB2-induced clustering. (A) Steady-state fluorescence anisotropy of ephrinB2-induced EphB2 clusters. COS-7 cells transiently expressing wtEphB2-mGFP or EphB2ΔSAM/PBM-mGFP were treated with 0.5 µg/ml unclustered ephrinB2-Fc. Anisotropy values of representative cells before (left) and 20 min after (right) stimulation with ephrinB2 are shown. Deletion of SAM and PBM domains leads to a decrease of fluorescence anisotropy (i.e., increase in clustering). Bars, 10 µM. The color coding of anisotropy values is shown on the right. (B) Quantification of steady-state fluorescence anisotropy plots from before and after stimulation (20 min) with 0.5 µg/ml of unclustered ephrinB2-Fc. Data represent mean anisotropy ± SEM of n = 35 and 46 cells for wtEphB2-mGFP and EphB2ΔSAM/PBM-mGFP, respectively. Post-stimulation curves are significantly different from each other; ***, P < 0.001; Mann-Whitney nonparametric test. (C, top) Representative Western blots of anti-Flag–immunoprecipitated wtEphB2 or EphB2ΔSAM/PBM (as indicated) after stimulation with equal concentration of Fc control or ephrinB2-Fc (0.5 µg/ml; t = 20 min) using mouse anti-phosphotyrosine antibody; blots were stripped and reblotted for total EphB2 protein levels. (bottom) Quantification of autophosphorylation (**, P < 0.01; ***, P < 0.001; unpaired t test; n = 7 separate experiments). (D) Quantification of collapse responses of HeLa cells expressing either wtEphB2 or EphB2ΔSAM/PBM induced by equal concentrations of unclustered human-Fc or ephrinB2-Fc (1 µg/ml is shown; similar results were obtained with 0.5 µg/ml). Cell collapse was scored by measuring cell surface area. Data are shown as mean cell area ± SEM from n = 25 cells per condition. Statistical significance was determined as in Fig. 4 A. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (wtEphB2 versus EphB2ΔSAM/PBM; n = 8 separate experiments). (E) Quantification of EphB2 surface proteins by immunostaining of individual wtEphB2 (n = 15 cells) and EphB2ΔSAM/PBM (n = 15 cells) transfected cells. Cells were fixed and stained with mouse anti-Flag antibody to visualize EphB2 expressed at cell membranes. Total EphB2 protein levels were visualized by YFP fluorescence. Ratios of surface/total fluorescence intensity integrated over the whole cell body were measured as an indication for expression levels at the cell surface. Scatter in range of EphB2 expression at the cell surface was similar for both conditions with a slight trend toward lower EphB2 level for EphB2ΔSAM/PBM-transfected cells. The experiment was repeated three times with the same outcome.

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