Contribution of EphrinB signaling to extracellular matrix-dependent assembly of HUVEC and MSC. (A-B) Flow cytometric detection of p-EphrinB in HUVEC and MSC transduced with wild-type EYFP-EphrinB2^WT (WT) or mutant EYFP-EphrimB2^5F (5F), with or without stimulation with EphB4-Fc (45 minutes). (B) Bottom reflects p-EphrinB and actin immunoblotting of MSC transduced with EYFP-EphrinB2^WT or mutant EYFP-EphrimB2^5F after sorting for YFP (> 90%) and stimulation with or without EphB4-Fc (45 minutes). (C) Matrigel-dependent cord formation by cocultures of YFP-sorted HUVEC and MSC transduced with EYFP-EphrinB2^WT (WT) or mutant EYFP-EphrimB2^5F (5F). WT reflects coculture of HUVEC and MSC transduced with EYFP-EphrinB2^WT (WT); 5F reflects coculture of HUVEC and MSC transduced with mutant EYFP-EphrimB2^5F (5F). (D) Western blot analysis of p-EphrinB and p-STAT3 expression in cocultures of HUVEC and MSC (ratio 2:1) incubated 1 and 3 hours in medium only or with PP2 onto Matrigel. The bar graph reflects relative ratios of p-EphrinB/EphrinB and p-STAT3/STAT3. (E) Representative images reflecting cord formation by HUVEC and MSC cocultured (ratio 2:1) on Matrigel for 1.5 hours. The bar graph reflects measurement of Matrigel-dependent network formation by HUVEC and MSC cultured together with or without PP2, as reflected by mean number of angles (± SD) generated by intersecting cords from 3 separate experiments; **P < .01. (F) Src silencing reduces EphB4-induced p-EphrinB in HUVEC and MSC. Western blot analysis of total Src and p-EphrinB in control (Risc-free siRNA) and Src-silenced HUVEC and MSC, with or without EphB4-Fc activation. The bar graph reflects relative ratios of Src/actin. (G) Effects of AG490 on p-EphrinB and p-STAT3 expression in cocultures of HUVEC and MSC (ratio 2:1) incubated onto Matrigel. Cells were incubated with AG490 on Matrigel with or without AG490 pretreatment (1-hour incubation with AG490). Results reflect immunoblotting with specific antibodies. (H) Jak2 silencing reduces EphB4-induced p-EphrinB in HUVEC. Western blot analysis of p-EphrinB, Jak2, EphrinB2, and actin in control (Risc-free siRNA), Jak2, or EphrinB2-silenced HUVEC, with or without EphB4 activation.