EphB-dependent activation of EphrinB in HUVEC and MSC incubated together on extracellular matrix. (A) Representative images showing p-EphrinB fluorescence immunostaining of HUVEC (unstained) and MSC (marked by red fluorescent dye PKH26red) cocultured on Matrigel. Bottom panels reflect images from bright-field microscopy. Original magnification ×20. (B) Time-dependent EphrinB activation in cocultures of HUVEC and MSC incubated on Matrigel detected by immunoblotting. (C) Flow cytometric detection of p-EphrinB in HUVEC (CD31⁺) and MSC (CD90⁺) removed from coculture on Matrigel at the indicated time points. The results reflect the means (± SD) of 3 independent experiments. (D) Representative images showing minimal p-EphrinB fluorescence in isolated cells from cocultures of HUVEC and MSC incubated 1 hour on Matrigel. Left panel: bright-field microscopy. Original magnification ×40. (E) Representative bright-field microscopy images from cocultures of HUVEC and MSC (ratio 2:1) incubated on Matrigel for 4 hours in the presence of the EphB receptor inhibitors SNEW and TNYL-RAW peptides. Original magnification ×4. (F) EphrinB2-Fc and peptides SNEW and TNYL-RAW reduce p-EphrinB in HUVEC and MSC incubated (2 hours) individually or together (ratio 2:1) on Matrigel. Representative immunoblotting results; the bar graph reflects the mean relative ratios (± SD) of p-EphrinB/total EphrinB2 band intensity detected by immunoblotting in 3 experiments. **P < .01; *P < .05. (G) p-STAT3 and STAT3 immunoblotting of cell lysates from HUVEC and MSC incubated (2 hours) together (ratio 2:1) on Matrigel in medium only (lane 1), with EphrinB2-Fc (lane 2), or with SNEW and TNYL-RAW peptides (lane 3). Results are from reprobing the membrane shown in panel E (right panel).