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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. 5. WT but not mutant efnB2/CTF2 forms complexes with VE-cadherin, Raf1 and Rok-α and increases angiogenic complexes.

Fig. 5

(A). Left: BAMECs were infected with HSV particles expressing efnB2/CTF2-myc3 or vector at 1MOI. 24h later cells were lysed, extracts were IPed with anti-Raf-1 antibodies and IPs were probed on WB for VE-cadherin (upper panel) or Raf-1 (middle panel). Extracts were also IPed with anti-myc antibody and efnB2/CTF2-myc3 was detected on WB with anti-ephrinB antibody (lower panel, lane 3). efnB2/CTF2-myc3 increases the Raf-1/VE-cadherin complexes (upper panel, lane 3). Right: Quantification of results. Paired t-test, n=4, **p < 0.01, error bars = S.E.M.

(B). Left: BAMECs were infected as in (A). Cell extracts were IPed with anti-Rok-α antibodies and IPs were probed on WB for VE-cadherin (upper panel) or Rok-α (middle panel). efnB2/CTF2-myc3 increases the Rok-α/VE-cadherin complexes (upper panel, lane 3) while N-cadherin/CTF2 (N-cad/CTF2) had no effect on these complexes (lane 4). Right: Quantification of results. Paired t-test, n=4, *p < 0.05, error bars = S.E.M.

(C). Left: Cells were transduced with pMX vectors expressing WT efnB2/CTF2-myc3 or mutants 5Tyr:Phe-myc3 or ΔPDZ-myc3. Cell extracts were IPed with anti-Raf-1 antibodies and obtained IPs were probed on WBs for VE-cadherin (upper panel) or Raf-1 (lower panel). WT efnB2/CTF2 increases the Raf-1/VE-cadherin complexes compared to vector pMX (lanes 4 and 5). Mutant efnB2/CTF2 peptides showed no increase (lanes 2, 3). Quantification of results. Paired t-test, n=4, **p < 0.01, error bars = S.E.M. Right: Extracts from the above cells were IPed with anti-VE-cadherin antibodies and IPs were probed for Raf-1 (upper panel) or VE-cadherin (lower panel) on WB. WT but not mutant efnB2/CTF2 increases the amount of Raf-1/VE-cadherin complexes (lanes 1–4). Quantification of results. Paired t-test, n=4, **p < 0.01, error bars = S.E.M.

(D). Left: Cells were transduced with WT efnB2/CTF2-myc3 or mutant ΔPDZ-myc3. Cell extracts were IPed with anti-Rok-α and IPs were probed on WBs for VE-cadherin (upper panel) or Rok-α (lower panel). WT but not mutant efnB2/CTF2 increases the Rok-α/VE-cadherin complexes. Right: Quantification of results. Paired t-test, n=4, *p < 0.05, **p < 0.01, error bars = S.E.M.

(E). Left: Cells were transduced with WT efnB2/CTF2-myc3 (CTF2-myc3) or mutants 5Tyr:Phe-myc3 or ΔPDZ-myc3. Cells were extracted and IPed as described in Figs 4 and 5. All three proteins, VE-cadherin, Raf-1 and Rok-α co-IPed with WT efnB2/CTF2 and to a much lesser extent with mutant peptides. Right: Quantification of results. Paired t-test, n=4, *p < 0.05, **p < 0.01, error bars = S.E.M.