Proteome profiler human apoptosis array kit

After treatment with TMZ for 24 hrs, the cells were harvested for analysis of apoptosis protein expression. The Proteome Profiler™ Human Apoptosis array kit (ARY009; R&D Systems) was used to detect the relative expression levels of 35 apoptosis-related proteins according to the manufacturer’s instructions. Briefly, ~1×10⁷ cells were solubilized in lysis buffer provided by the manufacturer. The recommended quantity of lysates was diluted and pipetted onto the membranes and incubated overnight at 2–8°C on a rocking platform shaker. Biotinylated secondary antibody cocktail provided by the manufacturer was pipetted onto membranes and incubated for 1 hr. After the washing process, the membranes were incubated with streptavidin-HRP provided by the manufacturer for 30 mins. The signals were developed using chemiluminescent reagents and then exposed to X-ray films. The positive signals were analyzed using ImageJ software.

A human apoptosis array was conducted using the Proteome Profiler Human Apoptosis Array Kit (ARY 009; R&D Systems, USA), according to the manufacturer’s instructions. Cells were lysed in protease inhibitor-added Lysis Buffer 17. After the arrays were blocked with Array Buffer 1 for an hour, they were incubated with the protein lysate at 4°C overnight, followed by washing and incubation with Detection Antibody cocktail (biotinylated antibody cocktail) for 1 h at room temperature. After completing the above steps, arrays were detected using Chemi Reagent Mix, and signals were captured using the ChemiDoc^TM XRS + Imager (Bio-Rad Laboratories).

The analysis was performed using Proteome Profiler™ Mouse Apoptosis Array Kit for C166 cells and Proteome Profiler™ Human Apoptosis Array Kit for lung fibroblasts, according to detailed protocols provided by the manufacturer (both kits from R&D Systems Inc., Minneapolis, MN). In brief, cells were lysed directly on the culture plates using Lysis buffer, provided in a kit, which was supplemented with a cocktail of proteinase inhibitors (Complete Mini, Roche Diagnostics, Mannheim, Germany). Cell lysates were vortexed and homogenized by their passing several times through a tip, and applied onto respective nitrocellulose membranes spotted with capture antibodies. After overnight incubation at 4°C, the membranes were washed and incubated with streptavidin-horseradish peroxidase conjugate. Then, stabilized luminol in a hydrogen peroxide solution was applied and membranes were scanned in the chemiluminescence detection system (FluorChem E system, ProteinSimple, San Jose, CA).
The signal density for each spot was measured using ImageJ software 1.51p (National Institutes of Health scientific image-analysis program) [9 (link), 10 (link)]. The relative expression of assessed proteins was shown as a percent of reference signal density, calculated as a ratio of mean signal density of the tested signal to mean density of reference spots. Each test was repeated twice.

The apoptosis-related proteins (including Bcl-2, Bax, Bad, pro-caspase-3, cleaved caspase-3, cytochrome C, TRAIL-R1/DR4, TRAIL-R2/DR5, FADD, Fas/TNFRSF6/CD95, HTRA2/Omi, p21/CIP1/CDKN1A, p27/Kip1, phospho-p53[S15], phospho-p53[S46], phospho-p53[S392], phospho-Rad17[S635], SMAC/Diablo, survivin, and TNF-R1/TNFRSF1A) were measured using the Proteome Profiler Human Apoptosis array kit (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer’s instructions. Tissue samples were homogenized in lysis buffer with a 1% protease inhibitor cocktail (Sigma-Aldrich Co., St Louis, MO, USA). After 30 minutes of incubation on ice, the protein concentration was quantified using the Coomassie (Bradford) Protein Assay Kit (PIERCE). Equal amounts of proteins (300 μg) from invasive and noninvasive tumor samples were incubated with the apoptosis array overnight at 4°C. After the washing of unbound proteins, a detection antibody cocktail was added to the membranes and incubated for 1 hour. Finally, streptavidin–horseradish peroxidase and Chemi Reagent Mix were used to reveal apoptosis-related proteins by chemiluminescence. Array images were scanned using MicroChemi 4.2 (Berthold Technologies, Chennai, India) and analyzed by ImageJ software.