Human cell stress array

Antibody array analysis was conducted using the Human Cell Stress Array (R&D Systems). For whole-cell extraction, 1 x 10 7 cells were harvested, washed with PBS, lysed in Lysis Buffer 6 supplemented with a proteinase inhibitor cocktail and a phosphatase inhibitor cocktail (Active Motif), incubated for 30 min on ice, and centrifuged at 4 C for 10 min at 15,000 rpm. Whole-cell lysates (1 ml) were mixed with 0.5 ml of Array Buffer 4 and 20 ml of reconstituted Detection Antibody Cocktail for 1 hr at room temperature. These samples were added to membranes blocked with Array Buffer 4. After incubation overnight at 4 C, the membranes were washed twice with 13 Wash Buffer and rinsed with distilled water and then dried. Diluted streptavidin-HRP (2 ml) was added and the membrane was incubated for 30 min at room temperature and then was washed with 13 Wash Buffer. Chemi Reagent Mix was applied evenly to the membrane and incubated for 1 min. Chemiluminescence signals were measured using LAS-3000 Imaging System (Fujifilm). Signal intensities were measured using Multi Gauge software. The ratios of signal intensities in cells treated with 40 mM APR-246 for 24 hr were calculated relative to the corresponding intensities in untreated cells.