cAng-1 induces Tie2 interaction with ILK on hypoxia, leading to stimulation of HIF-1α/SDF-1 axis. (A) Cells were transfected with pFlag-Tie2 plasmid and immunofluorescence stained with anti-Flag (green) and anti-ILK (red). Nucleus for TOPRO3 (blue). Confocal image showed the interaction of Tie2 and ILK as yellow color which was significantly enhanced at cell membrane by cAng-1. No fluorescence signal in the isotype IgG group. Magnification ×630 with immersion oil; scale bar, 20 μm. (B) Immunoprecipitation (IP) using anti-Tie2 followed by Western blotting for ILK. cAng-1 significantly increased the binding between Tie2 and ILK without change of total amount of ILK. WCL indicates whole cell lysate. (C-F) HUVECs were transfected with adenovirus-β-gal (Adv-β-gal) or kinase-deficient adenovirus ILK (Adv-ILK-dead-GFP) and treated with hypoxia alone or cAng-1 together. Augmentation of hypoxia-induced HIF-1α expression by cAng-1 was obliterated by ILK knockdown. (C) Western blotting and quantification graph of HIF-1α (n = 3). (D) HIF-1α reduction by dose-dependent transfection of Adv-ILK-dead-GFP (n = 2). (E) ChIP analysis shows that augmentation of HIF-1 binding to the SDF-1 promoter by cAng-1 was dependent on ILK (n = 2). (F) SDF-1 mRNA and quantification graph (n = 3). cAng-1–induced SDF-1 mRNA expression decreased in the ILK knockdown group.