Fig. 6. EphB4-Fc-induced sprouting and angiogenic complex formation depends on PS1. PS1 promotes angiogenic complexes in vivo.

(A). Cells were nucleofected with various concentrations of PS1 siRNA or buffer alone. After the indicated days, cells were lysed in 1% SDS lysis buffer and immunobloted with 33B10 mouse monoclonal antibodies against PS1/CTF and actin. Cortical neuronal extracts from PS1 WT and KO embryonic mice were used as a positive control for PS1.

(B). Cells were nucleofected with 7.5μM of bovine-specific PS1 siRNA or non-targeting siRNA. Cells were then used for the microcarrier bead assay in the presence of pre-clustered Fc or EphB4-Fc as described. The percentage of sprouts exceeding the diameter of the bead relative to control (Fc + non-targeting siRNA) was determined. Paired t-test (n=4, *p < 0.05, error bars = S.E.M.).

(C). Left: Cells were nucleofected with PS1 siRNA or control non targeting sequences as described in (B) and treated with 2μg/ml Fc or EphB4-Fc as indicated. Cell extracts were IPed with anti-Raf1 antibody and IPs were probed on WB for VE-cadherin (upper panel) or Raf-1 (middle panel). Input panel shows expression of VE-cadherin. Right: Quantification of results. Paired t-test, n=3, *p < 0.05, error bars = S.E.M.

(D). Left: Cell extracts from (C) were IPed with anti-Rok-α antbody and IPs were probed on WB for VE-cadherin (upper panel) or Rok-α (middle panel). Input panel shows expression of VE-cadherin. Right: Quantification of results. Paired t-test, n=3, *p < 0.05, error bars = S.E.M.

(E). Left: Brain extracts from WT or PS1 KO mouse embryos were IPed with anti-VE-cadherin antibodies and IPs were probed on WB for Rok-α (first panel) or VE-cadherin (second panel). Input shows Rok-α and VE-cadherin expression. Right: Quantification of results. Paired t-test, n=3, *p < 0.05, error bars = S.E.M.