Proteome profiler human array

To clarify the pathways through which DMC induced apoptosis, we determined apoptosis-related proteins using the Proteome Profiler Human Array (R&D Systems, Minneapolis, MN, USA). After treatment, cell lysate samples (200 μg) were extracted and applied per array set comprised of two nitrocellulose membranes with spotted capture antibodies. Next, a biotinylated antibody cocktail and streptavidin–horseradish peroxidase were used to detect the bound material using chemiluminescence detection. The pixel density of spots was quantified using Image-Pro Plus software. Spot densities were normalized against respective reference array spots and then against controls.

The supernatants were collected from the fibroblasts cultured with or without MPCs for 48 h, and were subjected to proteomic analyses using the Proteome Profiler Human Array (R&D, Cat. # ARY007) according to the manufacture's instruction. In brief, these samples were loaded onto the membrane spotted with the antibodies for specific markers. After overnight incubation, the results for different substrates were developed and detected. Quantification of the pixel densities was determined using AlphaEaseFC 4.0 software. Each spot was analyzed by Vision Works LS software (UVP, CA). The data were first normalized to positive control, and presented as the fold changes.

To identify the apoptosis-related proteins involved in FLLL32-induced apoptosis, the Proteome Profiler Human Array (R&D Systems, Minneapolis, MN, USA) was used for analysis. This kit can simultaneously detect 35 human apoptosis-related proteins. The Image-Pro Plus software was used to quantify the pixel density of the spots. The point density was normalized to the respective reference array points and then normalized to the control.