Phospho kinase array kit

Kinome profiling was performed using Phospho-Kinase Array Kit according to manufacturer’s protocol (R&D Systems, Oxford, UK). Pleural tissues from fiber-exposed and control mice were collected by dissection, homogenized and lysed, the lysates from the pleurae of 4 animals were pooled for each treatment group. Lysates, cleared by centrifugation, were loaded onto the provided membranes pre-coated with capture antibodies, and the presence of bound phospho-proteins was determined by western blotting with a mixture of detection antibodies. The signal intensities for kinase phosphorylation were determined in duplicate by densitometry and normalized on the provided positive control. The status of kinase phosphorylation in the pleurae of fiber-exposed animals is reported relative to the vehicle control.

We used a Phospho-Kinase Array Kit containing duplicate validated controls and capture antibodies that simultaneously detect the relative phosphorylation state of 43 human kinases (R&D Systems), according to the manufacturer's protocol. A total of 5×10⁶ cells were plated in 10 cm dishes and then deprived of serum for 24 h. In brief, the array membranes were blocked, incubated with 300 µg cell lysates per array overnight at 4°C. The next day arrays were washed three times, incubated with biotinylated antibodies for 2 h at room temperature, washed three times, incubated with streptavidin-HRP for 30 min at room temperature, washed again three times and developed with ECL western blotting detection reagents. The kinase spots were visualized with X-ray films (Ewen-Parker X-ray). The average pixel densities of duplicate spots were determined using the ImageJ software (http://imagej.nih.gov/ij/).

To further explore the mechanism of BUB1B in promoting CCA cell proliferation and invasiveness, the Phosphokinase Array Kit (ARY003B, R&D System, Minneapolis, MN) was employed to detect the relative levels of phosphorylation of 43 kinases. Cells were collected and lysed with a cell lysate buffer containing phosphatase and protease cocktail inhibitors (Roche, Mannheim, Germany), and then incubated at 4 °C for 15 min. After being centrifuged at 14,000 × g for 5 min, the supernatant was transferred into a clean test tube and the sample protein concentrations were quantified using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. The Array Detection steps were followed according to the manufacturer’s protocol. The sample protein (600 μg) was incubated with an antibody-array membrane at 4 °C overnight. The signals were detected and quantified using an Image Quant™ Imager (GE Healthcare Bioscience AB, Uppsala, Sweden) after the membranes were incubated with cocktail-detection antibody and streptavidin horseradish peroxidase.