Proteome profiler human phospho kinase array kit

We used a human proteome profiling array system (R&D Systems) that contains duplicate validated positive and negative controls and captures antibodies that can simultaneously detect the phosphorylation state of 43 human kinases (Proteome Profiler Human Phospho-Kinase Array Kit; for coordinate annotation, see https://www.rndsystems.com/; ref. 31 ). Five million cells were plated in 10-cm dishes and grown for 48 hours. Cells were deprived of serum by culturing for 24 hours in growth media supplemented with 0.05% FBS. Whole-cell extracts were then prepared, and detection of protein phosphorylation was carried out according to the manufacturer's instructions. In brief, the array membranes were blocked, incubated with 350 µg total cellular protein per array overnight at 4°C on a rocking platform, washed, and incubated with phospho-specific detection antibodies. Captured phosphorylated proteins were detected by ECL and imaged on X-ray films. The average pixel densities of duplicate spots were measured using ImageJ software and are expressed relative to the positive control on each array.

The Proteome Profiler Human Phospho-Kinase Array Kit and the Human Protease/Protease Inhibitor Array Kit were used according to the manufacturer's instructions (RnD Systems, Darmstadt, Germany), followed by densitometrical analysis with the Quantity one software (Biorad, Dreieich, Germany).