Angiostatin

The uterine samples were homogenized in a lysis buffer (Kurabo, Osaka, Japan). Then, the homogenates were centrifuged, and the resultant supernatants were collected. Western blotting was performed as described previously 3 (link). Briefly, membranes were incubated at room temperature for 1 h with primary antibodies against plasminogen (1 : 1,000; GeneTex, Hsinchu City, Taiwan), plasmin (1: 1,000; GeneTex), tPA (1:1000; Abcam), matrix metalloproteinase (MMP)-12 (1: 1,000; Proteintech Rosemont, IL, USA), angiostatin (1: 1,000; Abcam), or β-actin as a loading control (1 : 5,000; Sigma-Aldrich, St. Louis, MO, USA). The membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA). Immune complexes were detected using ImmunoStar Zeta reagent (Wako, Osaka, Japan), and images were acquired using Multi Gauge software (Fujifilm, Greenwood, SC, USA).

Specific ELISAs were used asper the manufacturers’ (all from R&D Systems unless otherwise stated) instructions to assess the plasma levels of TNF-α, granulocyte colony-stimulating factor (G-CSF), PDGF-BB, stromal derived factor-1 (SDF-1), total anti-oxidant capacity (Abcam), VEGF, endostatin, angiostatin (Abcam), thrombospondin-1, and thrombospondin-2.

Following gene transfection, cells were collected and lysed in ice-cold RIPA extraction solution (Thermo, Rockford, IL, USA). Cell proteins were separated by 8–15% SDS-PAGE and transferred to PVDF membranes using a Mini Trans-Blot Cell and System (Bio-Rad, Hercules, CA). The membranes were blocked with nonfat milk and incubated overnight with primary antibodies diluted in TBST at 4°C. The following primary antibodies were used: FILIP1L, angiostatin, endostatin, and matrix metalloproteinases (MMP)-2 and -9 (Abcam, Cambridge, UK); phospho-Akt (Ser473), Akt, phospho-β-catenin (Ser33/37/Thr41), β-catenin, phospho-glycogen synthase kinase 3 beta (GSK-3β) (Ser9), GSK-3β, cleaved caspase-3, -7, -9, and hypoxia-inducible factor-1α (HIF-1α) (Cell Signaling, Danvers, MA, USA); and vascular endothelial growth factor (VEGF)-A and -D, and GAPDH (Santa Cruz Biotechnology). The membranes were developed using an ECL reagent (Amersham, Arlington Heights, IL, USA). Immunoblots were quantified using Multi-Gauge software (ver 3.0, Fujifilm, Tokyo, Japan).