Human phosphokinase array

Cells were treated with or without necrotic cells for 24 h, and then lysed with array lysis buffer. Quantitation of protein concentration was performed using BCA assay. Cell lysates (430 μg of total proteins per array) were applied to the phosphoprotein array following the manufacturer’s protocol (Proteome Profiler^TM Arrays, R&D Systems). Block buffer was contacted into each membrane for 1 h on a rocking platform shaker. Cell lysates were diluted in array buffer and were incubated overnight at 4 °C. The array was incubated in each reconstituted detection antibody cocktail for 2 h at RT, then was diluted streptavidin-HRP for 30 min, followed by application of chemiluminescent reagent and expose to X-ray film (AGFA). The phosphokinase activity was measured by the changed spot intensity divided by reference spot intensity, which using Image J software.