Figure 4

Activation of STAT5 and Akt by TF/FVIIa interaction. A, western blot assays for expression of STAT5, STAT3 and Akt, as well as their phosphorylated products (p-STAT5, pSTAT3 and p-Akt) in TF-transfected SK-N-SH cells treated with FVIIa. Cells were incubated with or without 10 nM FVIIa for 10 min and 20 min respectively, and then cell extracts were analyzed for protein expression as indicated. B, graphical representation of the relative protein levels of p-STAT5, p-STAT3, and p-Akt compared to non-phosphorylated proteins in SK-N-SH/TF cells treated with FVIIa. Data are representatives of at least three independent experiments. C, phosphorylation of STAT5 by the interaction between FVIIa and either full-length TF or different truncated TF proteins. SK-N-SH cells transfected with different plasmids as indicated were incubated with 10 nM FVIIa for different time. Phosphorylation of STAT5 was detected by western blot assay. Data shown as the fold induction of mean (± SD) p-STAT5 levels from at least three independent experiments, compared to controls. The expression of either TF or HA (tag) in SK-N-SH cells transfected with different TF plasmids was detected by western blot (insert). The TF mAb (TF9-10H10, Calbiochem) only recognizes the extracellular domain of TF. D, Blockage of TF/FVIIa-induced phosphorylation of STAT5 and Akt by specific JAK and PI3K inhibitors, respectively. TF-transfected SK-N-SH cells were incubated with or without 10 nM FVIIa, along with either 600 nM JAK inhibitor 1 or 20 μM of the PI3K inhibitor LY294002 as indicated for 20 min. Induction of p-STAT5 and p-Akt as well as expression of STAT5 and Akt were detected by western blot assay.