Mouse angiogenesis array kit

Proteome profiler array was performed using the Mouse Angiogenesis Array Kit (R&D Systems) as per the manufacturer's instructions. Briefly, tissues were homogenized in PBS plus protease inhibitors and lysed with 1% triton X-100 and a freeze/thaw cycle. Equal amounts of protein lysates from three different tumours within each group were combined for experiments. Samples were incubated with provided membranes and proteins were visualized as described by the manufacturer. Proteins were quantified by measuring the mean pixel density of the individual spots and adjusted based on reference spots using the ImageJ software.

Protein was extracted using TRIzol Reagent (Thermo Fisher Scientific). Angiogenesis antibody array analysis was performed using the Mouse Angiogenesis Array kit (R&D Systems, ARY015; detecting 53 proteins) according to manufacturer's protocol. Briefly, 200 µg protein/membrane was hybridized on the antibody array membrane at 4°C overnight. A 1:5000 dilution of Streptavidin‐HRP was used for detection. Membranes were imaged using the Protein Simple FluorChem E system, and data were analyzed using ImageJ and normalized using internal controls. The experimental groups included: (1) HG, (2) HG + OGD, and (3) HG + OGD + 10 ng/ml VT.

Whole heart ventricles were snap-frozen in liquid nitrogen, and then grinded using a mortar and pestle. Approximately 20 mg of tissue per sample was used for further analysis. Tissues were incubated with 100 µL RIPA lysis buffer (65 mM Tris-HCl, 150 mM NaCl, 1% Triton-X-100, 1% Sodium deoxycholate, 1 mM EDTA, pH 7.4), and a mixture of protease inhibitors using cOmplete™ ULTRA Tablets (Roche, #5892791001). Lysates were kept on ice for 15 min, vortexed, and subsequently sonicated for three cycles (30 s ON, 30 s OFF) on low settings using a Bioruptor® Plus (Diagenode, #B01020001). Samples were then centrifuged for 10 min at 10,000 × g and supernatant was collected. A BCA protein assay kit (Thermo Scientific, #23225) was used to determine protein content of the supernatant fractions and 300 µg of total protein was used for the following steps. Angiogenic proteins were measured using a Mouse Angiogenesis Array Kit (R&D Systems, #ARY015) according to the manufacturer’s instructions. Digital images were quantified by densitometry using LI-COR Image Studio Lite software (version 5.2.5).

The assay was performed for the CM using a mouse angiogenesis array kit (R&D Systems, Inc.) according to the manufacturer’s instructions. VEGF-A production was examined by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Raybiotech, Atlanta, GA, USA) according to the manufacturer’s instructions.