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研究発表を行った学会；
・BMB2008 (第31回日本分子生物学会年会・第81回日本生化学会大会 合同大会）.
　２００８年１２月９日～１２日(神戸)

タイトル；Proteomic analysis for post-translational modification of transcription factors that control stem cell fate decision.
　
発表者；　新森　加納子　氏
　　　（熊本大学　発生医学研究センター　転写制御分野）
Abstract；
　The aim of this study is to identify proteins that serve as a switch regulating the states of undifferentiation and differentiation in neural stem cell (NSC). Especially, we focused on the changes of post translational modification of nuclear proteins such as transcription factors in response to stimulation by fibroblast growth factor 2 (FGF2), a representative factor to maintain neural stem/progenitor cells. Neuroepithelial cells from E14.5 mouse forebrain were cultured in FGF2-containing medium for five days to expand the NSC pool. NSCs were stimulated by FGF2 for 0 and 60 minutes after 6 hours of FGF2 starvation. Subsequently, NSCs were harvested, fractionated to enrich nuclear proteins, and subjected to the analysis by 2D-DIGE (two dimensional differential gel electrophoresis, 24 x 20cm gels with a pH range of 3-11) combined with a phospho-specific protein staining method. Four sets of 2 nuclear fractions (0min and 60min) were labeled with different fluorescent dyes (Cy3 and Cy5), mixed together with Cy2 labeled internal control (a pooled fraction from all of 8 samples) and separated in the identical 4 gels. The gels were scanned by a laser scanner and 12 images of 2D-PAGE were obtained from 4 sets of 2 samples. Simultaneously, to identify the specific phosphorylated protein spots, a pool of nuclear protein extracts was also separated by 2D-PAGE and stained with ProQ Diamond. After spot matching and profilings, statistical quantitative analysis of the protein patterns was carried out by using image analysis software, DeCyder. The result revealed that 15 up- or down-regulated protein spots in total 4095 spots were found with high significant differences in FGF2 stimulated nuclear fractions. Among them, we focused 10 spots differentially phosphorylated in response to FGF2, and identified all of them by nanoLC-QQTOF MS analysis. These proteins contain factors related to nuclear dynamics including nuclear translocation and modifications. Further functional analysis will give us an important information to elucidate the mechanism of on/off stem cell switching, and this sequential proteomic analysis system would be useful for finding molecules related to the neural stem cell fate decision.