Proteome profiler human phospho kinase array kit

The phosphorylation status of the cancer cell proteins was assessed using the Proteome Profiler Human Phospho-Kinase Array Kit (R&D, Minneapolis, MN, USA) recognizing 37 kinase phosphorylation sites and two related proteins. The kit was performed according to the manufacturer´s instructions and spots evaluated using the ImageJ and Origin software.

MACS-sorted CD8+ T cells were cultivated in the presence of DF- and MAF-CM as above. Subsequently, T cells were exposed to a-CD3 (HIT3a) and a-CD28 (CD28.2) for 30 min, on wet ice, in the original cell culture media, and then washed twice with ice-cold PBS and finally activated by adding AffiniPure F(abʹ)2 Fragment Goat Anti-Mouse IgG, F(ab')2 fragment-specific crosslinker antibody (Jackson ImmunoResearch, 115–006-006) in DMEM at 37 °C for 10 min. Cells were washed with ice-cold 1 × PBS, fixed and stained for flow cytometry, as above. In other experiments, cells were lysed and analyzed by a Proteome Profiler Human Phospho-Kinase Array Kit (R&D, ARY003B), as per the manufacturer’s instructions. Specific signals were detected by a ChemiDoc XRS + system (Bio-Rad), and normalized to Hsp60 as internal reference.

Cell lysates were assayed using a Proteome Profiler Human Phospho-Kinase Array kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Cells were grown in T25 flask until reaching 70–80% confluence and afterward treated with the appropriate concentration of β-ionone or solvent only (0.1% DMSO) for 10 or 30 min in a humidified incubator at 37°C. After a washing step with PBS^−/−, protein isolation was performed as described under “Western Blot.” Detection of relative phosphorylation levels of specific kinases was conducted with the Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems, Minneapolis, Minnesota, USA) according to manufacturer's protocol or by Western blot with phospho-specific antibodies according to manufacturer's instructions. Detection was done as described in Gelis et al. (2016 (link)). The protein levels were quantified using the Java-based ImageJ 1.46 software (Schneider et al., 2012 (link)) and the relative pixel intensities of odorant-stimulated samples were normalized to the relative pixel intensities of DMSO-treated samples.

The Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems) was used according to the manufacturer's instructions. The array membranes were incubated with 300μg cell lysate overnight. The detected signals were quantified using ImageLab software (BioRad). The signal of the negative control was subtracted from each spot, and values were normalized to the reference spots in each membrane.

Isolated human PBMCs were serum-deprived overnight and subsequently T cells were isolated to a purity of 95-99% by using the Pan T cell isolation kit. Purity and viability of the purified T cells were assessed by flow cytometry as for Western blot analysis of cyclins. Approximately 6x10⁶ serum-starved T cells were treated with 10 ng/ml of WT-rLifA or left untreated for 1 h followed by anti-CD3/anti-CD28 stimulation for 15 min in a 6-well plate containing a final volume of 2 ml cRPMI medium/well. Levels of human protein kinase phosphorylation was determined using the Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems). All procedures were performed according to the manufacturer’s instructions. Stimulated T cells were lysed and 150 µg of total protein lysates were applied to nitrocellulose membranes containing capture antibodies specific to protein kinases as a series of separate spots. Images were captured by ChemiDoc™ Gel Imaging System (BioRad). Pixel density in each spot of the array was quantified by using Image Lab software (BioRad). The relative signal intensity was expressed as fold-change relative to the untreated cells.

The phospho-antibody array analysis was performed using the Proteome Profiler Human Phospho-Kinase Array Kit from R&D Systems according to the manufacturer’s instructions. After a 24-h stimulation period, macrophages were lysed with Lysis Buffer 6 (R&D Systems) and agitated for 30 min at 4°C. Cell lysates were clarified by microcentrifugation at 14,000 × g for 5 min, and the supernatants were subjected to protein assay using a Pierce ™ BCA Protein Assay Kit (Thermo Scientific). Preblocked nitrocellulose membranes of the Human Phospho-Kinase arrays were incubated with ~400 μg (ES/whole parasite stimulation w/o LPS) or ~240 μg (stimulation with LPS) of cellular extract overnight at 4°C on a rocking platform. The membranes were washed three times with 1× Wash Buffer (R&D Systems) to remove the unbound proteins and were then incubated with a mixture of biotinylated detection antibodies and streptavidin-HRP antibodies. Chemiluminescent detection reagents were applied to detect spot densities. Membranes were exposed to X-ray film for 3, 5, and 10 min. Array images were analyzed using image analysis software Quantity One (Biorad).