4a–f) was highly reproducible. Fig. 2 3D-landscapes of selected gel areas. Relative spot intensities from controls (a, c, e) and RF-EME exposed cells (b, d, f) are depicted as spot heights to demonstrate the specific induction of some proteins relative to the local spot environment. The indicated proteins are also listed in Table 1 Fig. 3 Identification details of isolated 2D gel spots. After tryptic digestion and peptide P005091 in vitro separation by nano-flow
liquid chromatography, isolated peptides were fragmented in an ion trap mass spectrometer. a–c Peptides identified in the spot identified as ubiquitin carboxyl-terminal hydrolase 14 (z, peptide charge; Score, Spectrum Mill peptide score; SPI, scored peak intensity). b Assignment of identified peptides to protein sequence. c MS2 spectrum of the peptide AQLFALTGVQPAR. d–f Peptides identified in the spot identified as 26S protease
regulatory subunit 6B, e assignment of identified peptides to protein sequence, f MS2 spectrum of the peptide ENAPAIIFIDEIDAIATK Fig. 4 The RF-EME induced increase of 35S incorporation rates was reproducibly observed PI3K inhibitor in different cell types. a b and c, d show two independent experiments with Epigenetic Reader Domain inhibitor Jurkat cells. e, f is a representative example for cultured human fibroblasts showing the highest induction of 35S incorporation rates by RF-EME, g, h shows a representative example of quiescent (metabolically inactive) primary human white blood cells (WBC). Here, RF-EME hardly induced Niclosamide detectable increases in 35S incorporation rates; compared to untreated controls (g), activated WBC (i) displayed higher 35S incorporation rates, RF-EME induced a further increase in 35S incorporation rates (j), which indicates that activity renders cells sensitive to RF-EME Fibroblasts Cultured human fibroblasts showed the highest level of responsiveness to RF-EME (Fig. 4e, f; Table 2) with an average protein synthesis increase of 128 ± 22% (three independent experiments). Thirteen of the fourteen proteins whose rate of de novo synthesis was increased in Jurkat cells were also synthesized at a higher rate in fibroblasts. As well as these, the rates of synthesis of annexin
A1 and A5 were found to be significantly increased (Table 2). This finding suggests that the proteome alterations in responsive cells induced by RF-EME exposure are characteristic for this kind of cell stress. White blood cells Primary mononuclear cells isolated from peripheral blood (white blood cells, WBC) responded only marginally to RF-EME (Fig. 4g, h; Table 3). The apparent increase in 35S incorporation was less than 10%, which is within the margin of error of the applied methodology. Inflammatory stimulation of WBCs by treatment with lipopolysaccharide and phytohaemagglutinin increased the level of protein synthesis by these cells (compare Fig. 4g–i), which is consistent with the induction of cell proliferation as previously described in more detail (Traxler et al. 2004).