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Figure 7


Fig. 7. Translocation of the EphBR–ephrinB complex to inter-endothelial junctions prior to internalization. HUVECs that overexpress full-length ephrin-B2 or {Delta}C-ephrin-B2 were stimulated for various periods of time with EphB4-Fc after which monolayers were trypsinized, lyzed and processed for western blot analysis. (A) Ephrin-B2-overexpressing HUVECs presented ephrin-B2 on the cell surface. Trypsin treatment removed surface-expressed ephrin-B2 (lane 2 vs lane 1). Internalized ephrin-B2 was detectable within 15 minutes of EphB4-Fc stimulation. Maximal internalization of the EphB4–ephrin-B2 complex was detected after 30-60 minutes. Internalized ephrin-B2 was eventually degraded, as evidenced by the absence of ephrin-B2 after 4 hours. (B) By contrast, {Delta}C-ephrin-B2, albeit being correctly expressed at the cell surface (B, lane 2 vs lane 1), was not endocytoced following EphB4-Fc stimulation; an unspecific band was detectable at the same size as {Delta}C-ephrin-B2 (arrow in B). Yet, this band was also detectable in full-length ephrin-B2 overexpressing cells (arrow in A). (C) HUVECs that overexpress full-length ephrin-B2 or {Delta}C-ephrin-B2 were stimulated with EphB4-Fc for various periods of time after which monolayers were fixed and stained for ephrin-B2. Unstimulated HUVECs overexpressed ephrin-B2 uniformly on their cell surface (0 min). Stimulation with EphB4-Fc for 20 minutes led to the translocation of the resulting EphB4–ephrin-B2 and EphB4–{Delta}C-ephrin-B2 complex to inter-endothelial junctions (20 min). Complexes of full-length ephrin-B2 and EphB4-Fc were internalized within 60 minutes. By contrast, {Delta}C-ephrin-B2 complexes with EphB4-Fc were retained at inter-endothelial junctions (60 min). Analysis by fluorescence microscopy. (D) HUVECs that overexpress full-length ephrin-B2 or {Delta}C-ephrin-B2 were stimulated with EphB4-Fc for 30 minutes after which monolayer were fixed and stained for ephrin-B2 and VE-cadherin. Vascular integrity was not affected as evidenced by the unaltered junctional VE-cadherin expression. Ephrin-B2 did not colocalize with VE-cadherin; analysis by single scan confocal microscopy showing a single plain through the cell (notice the difference compared with images shown in C). Scale bars: 50 µm





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