Figure 7.

(A) TIRF and epifluorescence images showing the cluster formation of GFP-ephrinB2 WT or 4A. BT16 cells were transduced with lentivirus particles expressing GFP-ephrinB2 WT or 4A and plated on EphB4-Fc + laminin-coated well for 2 h. Scale bar, 5 µm. (B) Fluorescence intensities of ephrinB2 clusters were measured and averaged as described in Experimental Procedures. Error bars indicate the SEM of ephrinB2, n = 24; ephrinB2-4A, n = 16. The data are presented as the mean ± SEM. An unpaired two-tailed t test was used. *P < 0.05. (C) TIRF and epifluorescence images showing the cluster formation of GFP-ephrinB2 WT or 4A. BT16 cells were transduced with lentivirus particles expressing GFP-ephrinB2 WT or 4A and plated only on EphB4-Fc?coated well for 2 h. Scale bar, 5 µm. (D) Cell culture plates were coated with EphB4-Fc (5 µg/ml), and the free protein-binding sites were blocked with BSA. After cells were plated and incubated for the indicated times, the plates were washed, and quantitative cell adhesion assays were performed as described in Experimental Procedures. (E) Total protein expression of GFP-ephrinB2 WT or 4A in BT16 cells was verified by SDS?PAGE of a cell lysate followed by Western blot with anti-GFP antibody. (F) Cell surface protein expression on BT16 cells expressing GFP-ephrinB2 WT, 4A, or uninfected control was confirmed by flow cytometric analysis. Cells were harvested and stained with anti-GFP antibody and Alexa Fluor 647?conjugated secondary antibody.

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