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. 2015 Oct 26;291(5):2087–2106. doi: 10.1074/jbc.M115.696419

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — Ectopic expression of B-Raf(V600E) in WM35 enhanced gap formation by increasing TF expression and thrombin generation. A, WM35 cells were transfected with pBABE-bleo, pBABE(bleo)-FLAG-BRAF, or pBABE(bleo)-FLAG-BRAF(V600E) packed in retrovirus. Transfection efficiency was assessed by Western blotting with anti-FLAG and anti-B-Raf(V600E) antibodies. β-Tubulin serves as a loading control. B, WM35 cells transfected with pBABE-bleo, pBABE(bleo)-FLAG-BRAF, or pBABE(bleo)-FLAG-BRAF(V600E) were assessed for membrane TF expression. TF measurement is described in legend to Fig. 1B. *, p < 0.05 compared with pBABE(bleo)-FLAG-BRAF. C, WM35 cells transfected with pBABE-bleo, pBABE(bleo)-FLAG-BRAF, or pBABE(bleo)-FLAG-BRAF(V600E) were co-transfected with scrambled siRNA or siTF or treated with hirudin. They were co-cultured with HUVECs transfected with scrambled siRNA or siPAR-1 for 60 min in the presence of PFP before endothelial gap formation was measured. Values are mean ± S.E. from three independent experiments. *, p < 0.05. N.S., not significant.