cAng-1 up-regulates HIF-1α protein level and stimulates its binding on SDF-1 promoter. (A) Immunoblotting of HIF-1α by cAng-1 (200 ng/mL) under hypoxia at time-dependent (top) and quantification graphs (bottom) (n = 4, *P < .05). (B) Immunoblotting of HIF-1α by various concentration of cAng-1 under 6 hours hypoxia (top) and quantification graphs (n = 3, bottom; *P < .01). (C) Western blotting of HIF-1α in ischemic limb muscle. Induction of HIF-1α by ischemia was exaggerated and sustained by cAng-1 treatment (n = 3). (D) Tie2 siRNA (siTie2) abolished the effect of cAng-1 (200 ng/mL) on HIF-1α expression under 6 hours hypoxia (top). Quantification graph (bottom, n = 4). siCon indicates control siRNA. (E) ChIP analysis show HIF-1 binding to the SDF-1 promoter region. Lysates of HUVECs exposed to hypoxia for 6 hours were immunoprecipitated with antibody for HIF-1α. The precipitated DNAs were evaluated by PCR using specific primers for SDF-1 promoter (n = 3). (F-G) Silencing of HIF-1α in HUVECs abolished the effect of cAng-1 (200 ng/mL) on SDF-1 expression under hypoxia (*P < .01 vs Hypoxia; **P < .01 vs siCon).