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. Author manuscript; available in PMC: 2019 Aug 1.
Published in final edited form as: Cell Mol Life Sci. 2018 Feb 10;75(15):2813–2826. doi: 10.1007/s00018-018-2762-7

Fig. 4. EphB4-Fc increases the Raf-1/VE-cadherin and Rok-α/VE-cadherin complexes in a γ-secretase-and Rap1-dependent manner.

Fig. 4

(A). Left: BAMECs were treated with 2 μg/ml EphB4-Fc or Fc for the indicated times. Cells were lysed in 1%(v/v) TX-100 in the presence of protease inhibitors and extracts were IPed with anti-Raf-1 antibodies. IPs were probed on WB with anti-VE-cadherin (upper panel) or anti-Raf-1 (lower panel) antibodies. EphB4-Fc increases the Raf-1/VE-cadherin complexes (lanes 3, 6). Right: Bar graph shows quantification of results. Paired t-test, n=4, **p < 0.01, error bars = S.E.M.

(B). Left: Cells were treated with 2 μg/ml EphB4-Fc or Fc for 1h in the presence or absence of γ-secretase inhibitors L685,458 (1μM) or Compound E (1μM) added three hours prior to EphB4-Fc. Cells were lysed and IPed as in (A). EphB4-Fc-induced increase in Raf-1/VE-cadherin complexes (lane 2) was inhibited by both γ-secretase inhibitors (lanes 4,6). Right: Bar graph shows quantification of results. Paired t-test, n=4, **p < 0.01, error bars = S.E.M.

(C). Left: Cells were treated with 2μg/ml Fc or EphB4-Fc in the presence or absence of 0.5μM Rap1 inhibitor GGTi-298 as indicated in the figure. Cell extracts were IPed as in (A). Rap1 inhibitor strongly inhibits EphB4-Fc-induced complexes between Raf-1 and VE-cadherin. Right: Bar graph shows quantification of results. Paired t-test, n=4, ***p < 0.001, error bars = S.E.M.

(D). Left: Cells were treated with 2 μg/ml EphB4-Fc or Fc for the indicated times. Cells were lysed in TX-100 and extracts were IPed with anti-Rok-α antibodies. IPs were probed on WB with anti-VE-cadherin (upper panel) or anti-Rok-α (middle panel) antibodies. EphB4-Fc increases the VE-cadherin/Rok-α complexes (lanes 3 and 6) compared to Fc (lanes 2 and 5). Right: Bar graph shows quantification of results. Paired t-test, n=4, *p<0.05, **p < 0.01, error bars = S.E.M.

(E). Left: Cells were treated with γ-secretase inhibitors L685,458 (1μM) or Compound E (1μM) as above or DMSO for three hours and then treated with 2 μg/ml EphB4-Fc or Fc for 1h. Cell extracts were lysed and IPed as in (D). EphB4-Fc-induced Rok-α/VE-cadherin complexes (lane 2) were inhibited by both inhibitors (lanes 4,6). Right: Bar graph shows quantification of results. Paired t-test, n=4, *p < 0.05, error bars = S.E.M.

(F). Left: Cells were treated as in (C) and cell extracts were IPed as in (D). Rap1 inhibitor strongly inhibits EphB4-Fc-induced complexes between Rok-α and VE-cadherin. Right: Bar graph shows quantification of results. Paired t-test, n=4, **p < 0.01, error bars = S.E.M.