Profiling Phosphorylation Sites via Proteome Profiler

Relative protein phosphorylation levels were obtained by profiling 43 specific phosphorylation sites (Supplementary Table 4) using the Proteome Profiler Human Phosphokinase Array Kit (ARY003B, R&D Systems), according to the manufacturer's instructions. Briefly, the cells were rinsed with phosphate-buffered saline (PBS), resuspended in lysis buffer (1 × 10⁷ cells/mL), and agitated at 4°C for 30 min (29 (link)). The array membranes were blocked with 1.0 ml of Array Buffer one and incubated with 500 μg cell lysates at room temperature for 1 h. Next, cocktails of biotinylated detection antibodies were added and samples were incubated at room temperature for 2 h. Phosphorylated proteins were revealed using streptavidin-HRP/chemiluminescence substrate (sc-201696, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and autoradiography films. The resulting spots were scanned, and images were quantified using the Image Lab software (Bio-Rad).