Ab2904

The total protein of the tumour tissues was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) containing proteinase inhibitor cocktail, and the concentration of protein was determined using a bicinchoninic acid assay. Proteins (50 µg) were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (EMD Millipore, USA). The membranes were blocked with 5% skimmed milk at 4 °C overnight and incubated with primary antibodies against VEGFR2 (1:1,000; ab11939, Abcam), angiostatin (1:2,000; ab2904, Abcam), endostatin (1:500; ab202973, Abcam), Bcl-2 (1:1,000; ab32124, Abcam), and Bax (1:2,000; ab32503, Abcam) at 4 °C overnight. After incubating with sheep anti-rabbit IgG (1:5,000; ab97095; Abcam) at 37 °C for 1 h, protein bands were visualised using the enhanced chemiluminescence system (Thermo Fisher Scientific, Inc.). Protein expression levels were normalised to GAPDH expression (1:10,000; ab181602; Abcam) and quantified using ImageJ software version 1.46 (NIH).

The total protein extracts (20 µg) were separated on TGX 4%–20% precast gradient gels (Bio-Rad, Germany) and transferred onto nitrocellulose membranes. The membranes were blocked in 5% milk (for anti-angiostatin antibody) or 3% milk (for anti-VEGFα and anti-α-actinin antibodies). The membranes were then incubated with the following primary antibodies:
Polyclonal rabbit-a-angiostatin (Abcam, ab2904) 2 µg/ml in 5% milk, overnight at 4°C; monoclonal mouse-a-VEGFα (Abcam, ab1316), 5 µg/ml in tris-buffered saline (TBS)/T, 1 h at room temperature; rabbit-a-α-actinin (Cell Signaling, 3134), 1:1,000 in 5% milk, overnight at 4°C. After washing with TBS/T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (sheep anti-mouse, Jackson ImmunoResearch 515-035-003, 1:20,000 in 3% milk; goat anti-rabbit, Jackson ImmunoResearch JIM-111-035-003, 1:10,000 in 5% milk), washed again, and specific signals were detected using the ECL or Femto system (Thermo Fisher) according to the manufacturer's protocol. A densitometric analysis of the protein bands was done using ImageLab (Bio-Rad).

Paraffin sections (3 µm) of aortic vessels from WT and α-GAL-Tg/KO mice were deparaffinized and incubated in a 10 mM citrate buffer at 95°C for 40 min for antigen retrieval. Afterward, the sections were incubated for 10 min in 0.5% Triton/TBS for permeabilization, washed in TBS, and quenched in a 0.25% Sudan black solution for 30 min on a shaker in the dark. After repeated washing, the sections were blocked in 3% bovine serum albumin (BSA) in TBS for 1 h at room temperature. The sections were then incubated with primary antibodies [anti-angiostatin (Abcam ab2904), 1:20 in 3% BSA or anti-VEGFα (Abcam, ab1316), 1:400 in 3% BSA] overnight at 4°C, washed in TBS, and incubated with secondary antibodies [donkey anti-mouse Alexa Fluor 488 (A21202 life tech) or donkey anti-rabbit Alexa Fluor 488 (21206 life tech), 1:500 in 3% BSA] for 2 h at room temperature. After repeated washing in TBS, the slides were mounted with DAPI Fluoromount G (Biozol) and fluorescence signals were descriptively analyzed using a Zeiss Axiovert M200 with ApoTome.