Proteome profiler human xl oncology array ary026

A Human Oncology Array Kit (ARY026) was purchased from R&D (Minneapolis, United States). Briefly, the conditioned medium from untreated or ET-1-treated MCs (72 h) was added to each antibody-printed nitrocellulose membrane for overnight incubation at 4°C, following the manufacturer’s instructions. The membranes were visualized by using ChemiDoc Imaging System. Each pair of positive dots represented signals of highly expressed cytokines and the intensity was quantified by ImageJ software. The full list of all proteins candidates is available at the manufacturer’s official website (Proteome Profiler Human XL Oncology Array ARY026: R&D Systems (rndsystems.com).

A Human Cytokine Array Kit (ARY026) was purchased from R&D (Minneapolis, USA). Briefly, the conditioned medium was added into Array Buffers (R&D, ARY026) to a final volume of 1.5 mL and then applied to each antibody-printed nitrocellulose membrane for overnight incubation at 4 °C. On the next day, the membrane was incubated with the detection antibody cocktail (R&D, ARY026) for 1 h at room temperature. Treated with HRP-conjugated streptavidin antibody, the membrane was exposed to ChemiDoc. Each pair of positive dots represented signals of highly expressed cytokines and the intensity was quantified by ImageJ software. The full list of all proteins candidates is available at the manufacturer’s official website (Proteome Profiler Human XL Oncology Array ARY026: R&D Systems (rndsystems.com). Data extraction and analysis were performed after subtraction of the background and normalization to the internal references provided by the manufacturer, using an ImageJ protein array analyzer software (http://rsb.info.nih.gov/ij/macros/toolsets/Dot%20Blot%20Analyzer.txt).

The proteome was profiled using the Proteome Profiler Human XL Oncology Array ARY026 (R&D Systems, Minneapolis, MN, USA), which consists of 84 human cancer-related proteins. The HepG2 and Huh7 cells were treated with and without PNU-74654 (150 μM) for 24 h, and 200 μg protein was used for each matrix, according to the manufacturer’s instructions. A horseradish peroxidase-conjugated antibody was incubated with the membranes, followed by a chemiluminescent reagent, and the cells were examined with an ImageQuant LAS4000 instrument (GE Healthcare, Marlborough, MA, USA). ImageJ software was used to analyze the integrated density of each membrane [25 (link),26 (link)].