Ab97095

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).

Isolated histones were separated on a 4–20% SDS-PAGE gel and transferred to a PVDF membrane (iBlot^® 2 PVDF Mini stacks, Invitrogen) using the iBlot^® 2 dry blotting system on program P0 for 7 min. Membranes were blocked in TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) containing 5% milk powder and probed for H3K9ac (Abcam ab61231; 1:5000) or H4ac (K5 + K8 + K12 + K16 acetyl; Abcam ab10807; 1:5000) primary antibodies diluted in TBS-T. This was followed by anti-rabbit HRP (Abcam ab97095; 1:5000) secondary antibody and visualisation using Pierce SuperSignal^® West Pico chemiluminescent substrate.