Results Jurkat cells Figure 1 shows corresponding 2D gels derived from exposed and control cells. The separated proteins were q uantified for protein amounts (fluorescence detection) and protein synthesis (35S-autoradiography).
Screening Library The spot pattern obtained was very highly reproducible: 855 spots were consistently buy BGB324 detected in six gels from three independent experiments, each with exposed and corresponding control cells. The average standard deviation of fluorescence spot intensities (18.8%) was determined from the three independent control cell gels. Fluorescence spot intensities for some individual proteins appeared to reveal an increased level in response to RF-EME. Application of strict spot quantification criteria, however, indicated that there were no significant differences between RF-EME-exposed and control cells. Autoradiographs CHIR98014 order of the same gels, however, revealed significant differences in the rate of de novo synthesis of several proteins (greater than 2 fold) between RF-EME and control cells. Figure 1c, d shows the higher general autoradiograph intensity observed for radiation exposed cells. On average, the total 35S autoradiograph intensity
was almost doubled [the measured increase was 93 ± 28% (n = 3)]. Actually all detectable protein spots displayed an increased 35S incorporation rate. Fig. 1 RF-EME exposure of Jurkat T-cells generally increased 35S incorporation rates, indicating induction of protein synthesis. The cells (exposed and controls) were metabolically labeled for 8 h during exposure. a, b Fluorescence detection of protein amounts, separated by 2D-PAGE. c, d De novo synthesis (35S autoradiographs) of proteins depicted in a and b, respectively. While protein amounts did not show significant alterations,
protein synthesis was generally increased due to RF-EME. Annotated proteins are further detailed in Table 1 We categorized a protein as specifically up-regulated if the normalized integrated 35S autoradiograph spot intensity was at least two-fold greater than the corresponding control cell spot with an ANOVA P-value of oxyclozanide less than 0.05. Using this criterion, fourteen proteins were found to be specifically up-regulated and were subsequently identified by mass spectrometry as described in Materials and Methods (Table 1 and supplementary data). Figure 2 provides three examples of proteins specifically up-regulated by RF-EME: heat shock protein 70, ubiquitin carboxyl-terminal hydrolase 14 and 26S protease regulatory subunit 6B. Figure 3 shows peptide fragmentation mass spectra of peptides derived from ubiquitin carboxyl-terminal hydrolase 14 and 26S protease regulatory subunit 6 in order to demonstrate the high degree of reliability of the protein identification procedure used.