Proteome analysis of SYT-SSX protein complexes in synovial sarcomas.

为了解滑膜肉瘤发生的分子机制，我们采用保护酶法分析了滑膜肉瘤细胞中SYT-SSX蛋白复合物，检测了SYT-SSX蛋白复合物中的未知蛋白;我们将SYT、SSX或SYT-SSX cDNA表达质粒进行克隆，制备FLAG-lag融合蛋白和GST-tag融合蛋白。将这些质粒转染到HEK 293细胞中。制备细胞蛋白，用抗标签抗体珠将融合蛋白拉入细胞内，并进行蛋白质印迹分析。我们可以检测到FLAG标记的髓，但FLAG标记的SYT-SSX蛋白的yeti很低。在用GST标记的 *smiths进行的实验中观察到了几乎合理的结果，表明ION雪人对SYT-SSX融合蛋白在细胞核内的细胞定位是死亡的。因此，我们分离了细胞核，并在高癌和高盐条件下洗脱了核蛋白。将洗脱的核蛋白与细胞质蛋白混合以制备总细胞蛋白。从总细胞蛋白中纯化FLAG标记的SYT-SSX蛋白，并将膨化的FLAG标记的SYT-SSX蛋白温育并重构以制备SYT-SSX蛋白复合物。然后用FLAG-珠脉冲复合物，并将蛋白质应用于SDS-PAGE。用LC-MS分析了复合物的条带。SYT-SSX蛋白在某些抑制剂存在下的细胞定位分析。我们制备了SYT、SSX和SYT-SSX cDNA表达质粒以产生EGFP融合蛋白。将该质粒反向转染到我们建立的滑膜细胞系SYO-1中。我们发现SYTT-SSX蛋白以斑点状定位于细胞核内。我们建立了一个能在多西环素存在下表达SYT-SSX基因的诱导细胞系，并利用cDNA微阵列分析了诱导和非诱导细胞中SYT-SSX基因的表达模式。

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.