Proteome profiler human xl oncology array kit

After 24 h of incubation of A549 cells with or without ZER (50 µM) or ZER-LCNs (2.5 or 5 µM), the proteins were extracted with RIPA buffer ((Roche Diagnostics, Basel, Switzerland). The proteins were quantified by bicinchoninic acid (BCA) assay using Pierce BCA protein assay kit (Thermo Fisher). Three hundred micrograms protein per group was used to assess the expression of oncology-related proteins using proteome profiler human XL oncology array kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The protein signals in the array were photographed using the ChemDoc MP imaging system (Bio-Rad, Hercules, CA, USA). The Image J software (version 1.53c, Bethesda, MD, USA) was used to analyze the pixel densities of the protein signals.

Serum-free conditioned media were collected from cell lines and stimulated fibroblasts as described above and soluble factors analysed by cytokine array Proteome Profiler Human XL Oncology Array kit (R&D systems: ARY026) following the manufacturer's protocol. This kit provides relative expression levels for 84 human cancer-related proteins. Developed films were scanned at high resolution and analysed using ImageJ Studio software (Version 5.2; LI-COR, Inc, USA). Densitometry data were then normalised to the positive control spot (reference spots).

Relative protein expression levels of 84 human cancer-related proteins were analyzed by the Proteome Profiler Human XL Oncology Array Kit (Cat# ARY026; R&D systems, MN) according to the manufacturer’s recommendations. Briefly, membranes were placed in separate wells of the 4-well Multi-Dish with 2 mL of Array Buffer 6 for 1 h. Then, 200 μg total protein combined with Array Buffer 4 and Array Buffer 6 were added to the membranes and incubated overnight at 4 °C. On the next day, membranes were washed 3 times for 10 min with Washing Buffer solution and incubated with the Detection Antibody cocktail for 1 h. Subsequently, each of the membranes was washed 3 times and then incubated in 2 mL streptavidin-HRP for 30 min. The membranes were washed and covered with 1 mL Chemi Reagent Mix for 1 min and then exposed to an X-ray film in the dark at room temperature. For densitometric analysis, the Fiji software v1.51 (at https://imagej.net/software/fiji/) was used. Values were processed by WebMeV (Multiple Experiment Viewer at https://webmev.tm4.org/#/about) for visualization and stratification of data.