Streptavidin hrp chemiluminescence substrate sc 201696

Relative protein phosphorylation levels were obtained by profiling of 46 specific phosphorylation sites using the Proteome Profiler Human Phospho-Kinase Array Kit (ARY003B, R&D Systems). Experiments were performed according to the instructions provided by the manufacturer. Briefly, cells were rinsed with PBS, resuspended in lysis buffer (1×10⁷ cells per ml), and shaked at 4°C for 30 minutes. Membranes with spotted catcher antibodies were incubated with diluted whole cell lysates at room temperature for one hour. Next, cocktails of biotinylated detection antibodies were added and samples were incubated at room temperature for 2 hours. Phosphorylated proteins were revealed using streptavidin-HRP/chemiluminescence substrate (sc-201696, Santa Cruz biotechnology) and autoradiography films. The resulting spots were scanned and images were quantified using the Image lab software (Bio-Rad) and Microsoft Excel software.

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).