Tissue factor (TF) is both an initiator of blood coagulation and a signaling receptor. Using a proteomic approach, we investigated the role of TF in cell signaling when stimulated by its ligand, activated factor VII (FVIIa). From a 2-D difference gel electrophoresis (DIGE) study we found forty one spots that were differentially regulated over time in FVIIa stimulated cells or in comparison to nonstimulated cells. Mass spectrometry identifies 23 out of these as 13 different proteins. One of them, elongation factor 2 (EF-2), was investigated in greater detail by Western blot, a protein synthesis assay and cell cycle analysis. When tissue factor was stimulated by FVIIa, the phosphorylation of EF-2 increased which inactivates this protein. Analyzing the effect using site inactivated FVIIa (FVIIai), as well as the protease activated receptor 2 (PAR-2) agonist SLIGKV, indicated that the inactivation was not PAR-2 dependent. A panel of tissue factor mutants was analyzed further to try to pinpoint what part of the cytoplasmic domain that is needed for this effect. Performing a protein synthesis assay in two different cell lines we could confirm that protein synthesis decreased upon stimulation by FVIIa. Cell cycle analysis showed that FVIIa also promotes a higher degree of cell proliferation.
siRNA is a powerful tool in loss-of-function studies which generates valuable information on target validation. siRNA is highly selective and suppress gene expression in a sequence-specific manner. These features have made it a key tool in signal transduction - a cornerstone in cancer biology. 2-D DIGE (two dimensional differential gel electrophoresis) is a fluorescence based technology with the capacity to separate thousands of proteins in one single run, with high reproducibility. This makes it a valuable tool for differential expression analyses. In this study two powerful methods, siRNA and 2-D DIGE, were combined to investigate a well-known signaling pathway, pivotal in cancer biology. Silencing of the target protein made it possible not only to identify a large number of proteins that were differentially regulated but also to resolve the time dependency of protein regulation. Thus the combined use of siRNA and 2-D DIGE made it possible to find potentially new targets in the signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-276. doi:1538-7445.AM2012-LB-276
Background: The enzymatic mutation detection (EMD) assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves preformed heteroduplex molecules at mismatch sites, forming two shorter fragments that can be resolved by gel electrophoresis. The method can be used to detect single and multiple base changes, as well as insertions and deletions. Methods: The sensitivity, specificity, and positional accuracy of mutation detection by EMD with the PASSPORTTM Mutation Scanning Kit were assessed in a blind fashion for three analytical platforms (radioactive detection and automated laser sequencers ALFexpress and ABI PRISM 377). PCR products of 703 bp covering codons 188–393 of the P53 gene were prepared from colorectal tumor samples and analyzed by EMD; the results were compared to data from cDNA sequencing. A 1362-bp PCR product prepared from IL4r gene was used to test detection of multiple base changes in long PCR products. Results: The sensitivity for detection of mutations using EMD exceeded 90%, and the specificity exceeded 80% on all analysis platforms. The method localized 90% of mutations to within two codons and four codons for automated laser sequencers and detection by radioactivity, respectively. The method detected at least five mismatches in heteroduplexes >1 kb. Conclusions: The EMD system facilitates efficient detection of genetic variation in fragments exceeding 1 kb irrespective of location and type. The technology is particularly well suited to the detection of mutations in genes frequently mutated at unpredictable locations.
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