| Budget Amount *help |
¥3,730,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥330,000)
Fiscal Year 2007: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2006: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
In order to understand the molecular mechanism underlying the onset of synovial sarcomas, we analyzed fit SYT-SSX protein complex in synovial sarcoma cells by protecmics methods.
Detection of the unknown proteins the SYT-SSX protein complex;
We male the SYT, SSX, or SYT-SSX cDNA expression plasmid to produce FLAG-lag fusion protein and GST-tag fusion protein. These plasmids were transfected into HEK293 cells. The cellular proteins were prepared, and the fusion proteins were pulled dorm by anti-tag antibody-beads, and analyzed by western blotting. We could detect tie FLAG-tagged piths, but The yeti of FLAG-tagged SYT-SSX protein was low. The almost sane result was observed in tie experiment with GST-tagged *smiths, suggesting that the ION yeti was die to tie cellular localization of SYT-SSX fusion proteins within nucleus. So, we isolated the nucleus and the nuclear proteins were eluted in high cancer &fled salt condition. The eluted nuclear proteins were mixed will cytoplasmic proteins to make total cellular protein. FLAG-lagged SYT-,SSX protein was purified from the total cellular proteins, and the puffed FLAG-tagged SYT-SSX protein was incubated and re-constructed to make the SYT-SSX protein complex. Then the complex was puled by FLAG-beads, and the proteins were applied on SDS-PAGE. We analyzed sane bands of the complex by LC-MS.
Cellular localization analysis of SYT-SSX protein in the presence of some inhibitors. We made the SYT, SSX, and SYT-SSX cDNA expression plasmids to produce EGFP-fusion proteins. The plasmids were reverse-transfected into SYO-1 that is a synivial cell line we established. We found that the SYTT-SSX proteins localize in nucleus with speckled form. We analyzed the effect of some inhibitors on the localization of the SYT-SSX proteins.
We established an inducible cell line that can express the SYT-SSX gene in the presence of doxicyclin, and the gene expression pattern in the induced/non-induced cells were analyzed on cDNA microarray analysis.
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