Proteome profiler human phospho kinase array kit ary003

HCC cells were treated with WA and the phospho-antibody array analysis was performed using the Proteome Profiler Human Phospho-Kinase Array Kit ARY003 from R&D Systems (Minneapolis, MA) according to the manufacturer’s instructions. Array images were analyzed using the GeneTools image analysis software (Syngene).

Relative protein phosphorylation levels of 38 selected proteins were obtained by analysis of 46 specific phosphorylation sites using the Proteome Profiler Human Phospho-Kinase Array Kit ARY003 (R&D Systems, Minneapolis, MN, USA) in duplicate tests according to the manufacturer’s instructions. Briefly, cells were rinsed with PBS, 1 × 10⁷ cells/mL lysis buffer were solubilized under permanent shaking at 4 °C for 30 min, and aliquots of the lysates were stored frozen at −80 °C. After blocking, membranes with spotted catcher antibodies were incubated with diluted cell lysates at 4 °C overnight. Thereafter, cocktails of biotinylated detection antibodies were added at room temperature for 2 h. Phosphorylated proteins were revealed using streptavidin-HRP/chemiluminescence substrate (SuperSignal West Pico, Thermo Fisher Scientific, Rockford, IL, USA) and detection with a Molecular Imager VersaDoc MP imaging system (Bio-Rad, Hercules, CA, USA). Images were quantified using the ImageQuant TL v2005 software (Amersham Biosciences, Buckinghamshire, UK) and Microsoft Excel software (Microsoft, Redmond, WA, USA). The different Western blot membranes were normalized using the 3 calibration spots included. Signaling pathways affected by fascaplysin in NCI-H526 and A549 lung cancer cells were produced using Power Point software (Microsoft, Redmond, WA, USA).

Breast cancer cells were treated with 5 μM HNK and the phospho-antibody array analysis was performed using the Proteome Profiler Human Phospho-Kinase Array Kit ARY003 from R&D Systems (Minneapolis, MN) according to the manufacturer's instructions. Array images were analyzed using the GeneTools image analysis software (Syngene, Frederick MD).

Phosphokinase analysis was performed using the Proteome Profiler Human Phospho-Kinase Array Kit ARY003 (R&D Systems)^{53 (link)}. Array images were analyzed using the GeneTools image analysis software (Syngene). For Immunofluorescence, breast cancer cells (5 × 103) were seeded on chambered slides, allowed to grow for 24 h and treated as indicated and subjected to immunofluorescence analysis^{34 (link)}. Immunofluorescently stained cells were imaged using a Zeiss LSM510 Meta (Zeiss, Dublin, California, USA) laser scanning confocal system configured to a Zeiss Axioplan 2 upright microscope (Zeiss, Dublin, CA, USA).

Relative protein phosphorylation levels of 38 selected proteins in scrambled control-siRNA transfected S2-013 cells and CCDC88A-siRNA transfected S2-013 cells were obtained by profiling 46 specific phosphorylation sites using the Proteome Profiler Human Phospho-Kinase Array Kit ARY003 from R&D Systems (Minneapolis, MN), according to the manufacturer’s instructions. Briefly, cells were rinsed with PBS, and 1 × 10⁷ cells/ml lysis buffer were solubilized with permanent shaking at 4 °C for 30 min. Aliquots of the lysates were stored frozen at −80 °C. Membranes with spotted catcher antibodies were incubated with diluted cell lysates at 4 °C overnight. Thereafter, cocktails of biotinylated detection antibodies were added at room temperature for 2 h. Phosphorylated proteins were detected using a horseradish peroxidase conjugated anti-mouse/rabbit antibody. Blots were then incubated with an enhanced chemiluminescence solution for 1 min for signal detection.