Proteome profiler human cell stress array

The relative protein expression levels of a panel of 35 proteins related to apoptosis and 26 proteins related to cellular stress were obtained while using the Proteome Profiler Human Apoptosis Array (R&Dsystems- #ARY009) and Proteome Profiler Human Cell Stress Array (R&Dsystems-#ARY018), according to the manufacturer’s instructions and as previously reported [54 (link)]. The selected cell lines (GAMG and U373) were treated with IngC for 6 and 24 h, for stress array and 24 and 72 h for apoptosis array while using a concentration equivalent to 3 x IC₅₀ value of each glioma cell line. In addition, THE expression of some of the proteins was validated by western-blot analysis as described below.

Cell culture supernatants and lysates were centrifuged at 500 g for 5 min, and aliquots were stored at − 80 C until further use. Samples from two biologically distinct experiments were pooled for analysis, and each experiment was conducted using 3 technical replicates of samples. Angiogenic factors were measured in the cell supernatant using the Proteome Profiler™ Human Angiogenesis Antibody Array, and cell stress markers were measured in cell lysates using the Proteome Profiler™ Human Cell Stress Array (R&D system, USA) according to the manufacturer’s protocols. Briefly, 500 ml of supernatant or 150 mg of cell lysate was incubated with a cocktail of biotinylated detection antibodies and added to the array membrane. Following incubation on a rocker at 4 °C overnight, capture antibodies spotted in duplicate on the array were visualized using chemiluminescent detection reagents. The signal produced by each spot is proportional to the amount of bound analyte. The mean pixel intensity of the duplicate spots on the membrane was calculated and averaged using ImageJ Software.