2005; Schwarz et al. 2008). The exposure was 2 W/kg during the “on” phase. With the chosen parameters, the controlled temperature difference between the two chambers (control and real exposure) was below 0.15°C, which, according to our experience, excludes a thermally induced effect in our system (Gerner et al. 2002). Cell preparation Human Jurkat T-cells were cultured in RPMI supplemented

with 10% FCS under standard cell culture conditions. Primary human diploid fibroblasts (ES1 cells) were a kind gift of the workgroup Rüdiger in Vienna. It allowed us to investigate the proteomes of the very same cell line and culture conditions, which upon radiation revealed DNA breaks (Diem et al. 2005; Schwarz www.selleckchem.com/products/pf-06463922.html et al. 2008). These cells were cultured in Dulbecco`s

modified Eagle`s Medium (DMEM, Gibco), 10,000 IU/ml buy Wortmannin penicillin/streptomycin, 200 mM l-glutamine, 40 μg/ml neomycin and 10% FCS. Peripheral blood mononuclear cells (white blood cells—WBC) were isolated from heparinized whole blood obtained from healthy donors (mixed with 2 vol. HBSS) by standard density gradient centrifugation with Ficoll-Paque (Pharmacia Biotech). The interface cells were washed and resuspended in autologous (donor) plasma. Inflammatory activation of the cells was accomplished by the addition of 5 μg/mL phytohaemagglutinin (PHA-P; Sigma) and Selleck MS 275 10 ng/mL LPS (Sigma). Cells were metabolically labeled with 0.2 mCi/mL 35S protein labeling mix containing 35S-methionine and 35S-cysteine (Trans35label, Biomedica, MP Biomedicals) during control exposure and real RF-EME at 37°C in a humidified atmosphere containing 5% CO2. The incubation and labeling times were 2 and 4 h in exploratory experiments and 8 h in the final series with three independent repetitions per exposure condition. Subcellular fractionation After incubation and labeling of cells, cytoplasmic proteins were isolated Tyrosine-protein kinase BLK as follows. Cells were lysed in 0.25 M sucrose, 3 mM MgCl2, 0.5% Triton X-100 in lysis buffer (10 mM HEPES/NaOH, pH 7.4, 10 mM NaCl, 3 mM MgCl2). The cytoplasmic fraction was separated from the nuclei by centrifugation through a 30% sucrose gradient at 3,500 rpm for 5 min at 4°C. After ethanol precipitation,

the pelleted cytoplasmic protein fraction was directly solubilized in sample buffer. All buffers used were supplemented with the protease inhibitors PMSF (1 mM), aprotinin, leupeptin and pepstatin A (all at 1 μg/mL). 2D Page High-resolution 2D gel electrophoresis was carried out as described previously (Gerner et al. 2002), using the Protean II xi electrophoresis system (Bio-Rad, Hercules, CA). The protein samples were dissolved in sample buffer (7.5 M urea, 1.5 M Thiourea, 4% CHAPS, 0.05% SDS, 100 mM DTT). To optimize the solubilization of proteins, we saturated the protein solution with solid urea. Protein concentrations were determined using a standard Bradford assay. Solubilized protein (300 μg per gel) was diluted to 280 μl with sample buffer freshly adjusted to 0.