Fig. 4

Inhibition of TIME cells adhesion and phosphorylation of Tie2, Akt, and FAK. a 5 × 10⁴ TIME cells were incubated alone (cells only; black), with 500 ng/ml of FL-Ang1 (gray), or with a combination of 500 ng/ml FL-Ang1 and 1 μM cRGD (purple), Ang2-BD_WT (blue), Ang2-BD_BC5 (green), Ang2-BD_BC6 (yellow), or Ang2-BD_BC10 (red) for 2 h on vitronectin-coated 96-well plates. b 5 × 10⁴ TIME cells were incubated alone (cells only; black) or with 1 μM cRGD (purple), Ang2-BD_WT (blue), Ang2-BD_BC5 (green), Ang2-BD_BC6 (yellow), or Ang2-BD_BC10 (red) for 2 h on vitronectin-coated 96-well plates. c For determining Tie2 phosphorylation, TIME cells were treated with control buffer (basal level; black), 500 ng/ml FL-Ang1 (gray), or a combination of 500 ng/ml FL-Ang1 with 1 μM cRGD (purple), Ang2-BD_WT (blue), Ang2-BD_BC5 (green), Ang2-BD_BC6 (yellow), or Ang2-BD_BC10 (red) for 15 min on vitronectin-coated 12-well plates. d Cell lysates were analyzed by Western blot using antibodies against phosphorylated Tie2 (pTie2), Tie2, and β-actin. e Akt phosphorylation was determined as in c except that the cells were treated with a combination of 500 ng/ml FL-Ang1 with 0.5 μM of the above proteins and the incubation time was 30 min. f Cell lysates were analyzed by Western blot using antibodies against phosphorylated Akt (pAkt), Akt, and β-actin. g FAK phosphorylation was determined as in c. h Cell lysates were analyzed by Western blot using antibodies against phosphorylated FAK (pFAK), FAK, and β-actin. “*” indicates a P value < 0.05 upon comparing the results between the cells-only control (b) and the tested proteins. “*” indicates a P value < 0.05 upon comparing the results between FL-Ang1 alone (a, c, e, h) and a combination of FL-Ang1 with the tested proteins. “&” indicates a P value < 0.05 upon comparing the results between cRGD (a, b, c, e, h) and the tested proteins. “#” indicates a P value < 0.05 upon comparing the results between Ang2-BD_WT (a, b, c, e, h) and the tested proteins