Identification of MDH2 as a binding protein to angiostatin. Mouse liver homogenate was first passed over an mFc-Sepharose column followed by mFc-angiostatin-Sepharose step. PAGE analysis of the eluant is shown in panel A (arrow points to the major band identified as MDH2) using Coomassie stain. ELISA (B) was performed by coating the plate with porcine heart mitochondria MDH (1 μg/mL). The ligands are indicated at the bottom (1 μg/mL). (C) Either ATP synthase knocked down or wild-type tumor cells were transfected with GFP-MDH2 plasmid (transfection efficiency was ∼ 40%). Cells were permeabilized and incubated with hFcAS and anti-ATP synthase β monoclonal antibody. Alexa 568 antihuman and 647 antimouse antibodies were used for detection. HFcAS (red) showed colocalization with ATP synthase (pink) in ATP synthase wild-type cells and also with MDH2 (green) in ATP synthase knocked down cells. Bar represents 20 μm. (D) ATP production in HUVECs was inhibited by hFcAS in both extracellular (ExtraC) and intracellular (IntraC) regions. The incubation times were 30, 90, and 360 minutes. (E) A diagram summarizing the identified pathways after angiostatin treatments in vivo and in vitro.