The expression of angiogenesis-related proteins of ZER-treated HepG2 cells was evaluated by a semiquantitative technique (Proteome Profiler™, Human Angiogenesis Array Kit, RandD Systems, USA) according to the manufacturer's instructions. Duplicate spots on the nitrocellulose membranes were excised and the samples diluted and mixed with a cocktail of biotinylated detection antibodies. The protein components of the mixture was determined by Human Angiogenesis Array kit (Proteome Profiler™, Human Angiogenesis Array Kit, RandD Systems, USA). The protein-antibody complexes present were bound to the membrane cognate-immobilized capture antibody. After washing to remove unbound materials, streptavidin-HRP and chemiluminescent detection reagents were sequentially added. The light intensity of the spots is proportional to the amount of bound analyte. The image of the spots was captured on X-ray films and analyzed using Image J software (version 1.46d; US National Institutes of Health [NIH]). The results are expressed as fold changes above or below the unexposed cell cultures. Only proteins of interest were quantified and reported.
Proteome profiler human angiogenesis array kit
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Proteome Profiler Human Angiogenesis Array Kit is a multiplex array that allows for the simultaneous detection and quantification of 55 different human angiogenesis-related proteins in a single experiment. The kit includes a membrane with pre-spotted capture antibodies and detection antibodies for the target proteins.
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63 protocols using proteome profiler human angiogenesis array kit
1
Angiogenic Protein Expression in ZER-Treated HepG2 Cells
2
Quantitative Angiogenesis Secretome Analysis
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The manufacturer’s instructions analyzed the Secretome with the Proteome ProfilerTM Human Angiogenesis Array Kit (R&D System, Minneapolis, MN, USA). Quantification of secretome-containing proteins was performed via standard densitometry using Quantity One Software 4.4 (Bio-Rad, Hercules, CA, USA). Differential expression analysis was performed using the R programming environment (version 3.4.4). The dataset contained 55 different capture antibodies and two donors by cells (hMSC and hMSC-EC). The log2 and mean for each protein per group and the fold change between the two groups were calculated. For protein analysis, 0.5 μg/mL of total protein was used.
3
Angiogenic Potential of Extracellular Vesicles
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The angiogenic content of lysed DPSC and BM-MSC EVs and their corresponding EV-depleted CM was screened with the Proteome ProfilerTM Human Angiogenesis Array Kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol. The membranes were incubated with 1 mL of lysed EVs and EV-depleted CM concentrated from 25 mL CM of three different donors per cell type. Signal density was visualized and quantified by ImageQuant LAS 4000 Mini equipped with ImageQuant TL software. For every protein, signals of the negative control spots were subtracted and the corrected signal densities were normalized to the positive reference spots. The resulting relative pixel densities were represented in a heat map.
4
Profiling Glioblastoma-Microglia Secretome
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Cell culture media was collected from the following group: GBM-MG co-cultures (co-culture), GBM cells alone (GBM single), or MG cells alone (MG single). The media were spun down (300×g, 10 min) to remove any debris. As an additional control, we created a 1:1 mixture of GBM single and MG single media (Mix) that would not account for any GBM-MG crosstalk mediated shifts in secretome. The secretome for each specimen was profiled using a Proteome ProfilerTM Human Angiogenesis Array Kit (Ary007, R&D Systems, Minneapolis, MN) following the manufacturer’s protocol. Blots were imaged using an Image Quant LAS 4010 chemiluminescence imager (GE Healthcare). Dot intensities were quantified with the ImageJ macros toolset Protein Array Analyzer (Table S1 ). Data was first normalized by dividing the pixel intensity for each blot by the average positive control pixel intensity (on each membrane). We calculated intensity fold change between co-culture and mix groups, identifying factors that displayed a larger than 1.5-fold change; targets showing > 0.75-fold change relative to positive reference spot intensities was plotted.
5
Differential Angiogenesis Profiling of PAK1
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Proteome ProfilerTM Human Angiogenesis Array Kit (R&D, ARY007) was applied to search for differentially expressed angiogenesis-regulated molecules between shPAK1 and shLacZ transductions for both PAK1-overexpreassing OH931 and NMFH-1 cell lines. According to manufacture's instruction, the angiogenesis antibody membrane, spotted with 55 immobilized angiogenesis-associated antibodies in duplicate, was processed as detailed in Method-S4.
6
Profiling GBM-Microglia Secretome Interactions
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Cell culture media was collected from the following group: GBM-MG co-cultures (co-culture), GBM cells alone (GBM single), or MG cells alone (MG single). The media were spun down (300 x g, 10 minutes) to remove any debris. As an additional control, we created a 1:1 mixture of GBM single and MG single media (Mix) that would not account for any GBM-MG crosstalk mediated shifts in secretome. The secretome for each specimen was profiled using a Proteome Profiler TM Human Angiogenesis Array Kit (Ary007, R&D Systems, Minneapolis, MN) following the manufacturer's protocol. Blots were imaged using an Image Quant LAS 4010 chemiluminescence imager (GE Healthcare). Dot intensities were quantified with the ImageJ macros toolset Protein Array Analyzer (https://imagej.nih.gov/ij/macros/toolsets/Protein%20Array% 20Analyzer.txt, Gilles Carpentier) (Table S1). Data was first normalized by dividing the pixel intensity for each blot by the average positive control pixel intensity (on each membrane). We calculated intensity fold change between co-culture and mix groups, identifying factors that displayed a larger than 1.5-fold change; targets showing >0.75-fold change relative to positive reference spot intensities was plotted.
7
Quantifying Angiogenesis Proteins in EVs
8
Characterization of Paracrine Factors from Adipose Stem Cells
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To characterize the paracrine activity of ASCs, conditioned medium was collected, centrifuged at 2,500 g to remove cell debris, and stored at –70 °C until the measurements were taken. To detect secreted proteins, conditioned medium was analyzed using the Proteome Profiler Human Angiogenesis Array Kit (R&D, USA) and Proteome Profiler Human Protease Array Kit (R&D, USA), according to the manufacturer’s instructions. The data were analyzed using Image Lab™ Software Version 5.0 (Bio-Rad, USA). VEGF-α, TGF-β, IL-6, IL-8, MCP-1, and IGF-1 concentrations in ASC conditioned medium were evaluated using the Human VEGF ELISA Set (Peprotech, USA), Human TGF-β1 DuoSet ELISA (R&D, USA), Human IL-6 ELISA Set (BD, USA), Human IL-8 ELISA Set (BD, USA), Human CCL2/MCP-1 DuoSet (R&D, USA), and Human IGF-1 DuoSet (R&D, USA), according to the manufacturer’s instructions.
9
Diabetic ssEVs Impair MVEC Angiogenic Factors
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To explore whether diabetic ssEVs impair secretion of pro‐angiogenic factors in MVECs, we examined angiogenic factors in conditioned culture medium in HMVECs using Proteome Profiler Human Angiogenesis Array Kit (Cat. #: ARY007, R&D System) according to the manufacturer’s protocol. The HMVECs were treated with ssEVs from either db/+ or db/db mice for 48 hours. To explore the role of EZH2 in diabetic ssEVs‐impaired secretion of pro‐angiogenic factors, HMVECs were treated with diabetic ssEVs plus EZH2 specific inhibitor GSK343 (0.1 µM) for 48 hours. Quantification of selected angiogenic factors in culture medium of HMVEC was carried out by densitometry using ImageJ.
10
Angiogenic Protein Detection in Cell Supernatants
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