Project description:EVs in culture supernatant can be concentrated with removing exsomeres and 97% free proteins by MWCO 750 kDa UF. DEAE chromatography can be divided into bioactive EXO and other EVs as nucleic acid (DNA) cargo in UF-concentrated EVs.
Project description:EVs in culture supernatant can be concentrated with removing exsomeres and 97% free proteins by MWCO 750 kDa UF. DEAE chromatography can be divided into bioactive EXO and other EVs as nucleic acid (DNA) cargo in UF-concentrated EVs.
Project description:EVs in culture supernatant can be concentrated with removing exsomeres and 97% free proteins by MWCO 750 kDa UF. DEAE chromatography can be divided into bioactive EXO and other EVs as nucleic acid (DNA) cargo in UF-concentrated EVs.
Project description:Gene expression analysis of WT and IL-2Ra-deficient CTL (P14) isolated 8 days after inffection with LCMV. The goals of the study are to assess the impact of IL-2 signals on effector and memory CTL differentiation.
Project description:In cytotoxic T cells (CTL), Protein Kinase B /Akt is activated by the T cell antigen receptor (TCR) and the cytokine Interleukin 2 (IL2), in part by phosophorylation of Akt by Phospholipid dependent kinase 1 (PDK1). The role of PDK1 and Akt in CTL has however not been fully defined. In order to explore the relative roles of these kinases in CTL we used microarrays to profile the gene expression of control and PDK1 null CTL. In separate experiments we compared the gene expression profiles of control and Akt inhibitor treated CTL. CTL were generated from 3 mice each carrying two loxP flanked PDK1 alleles plus a tamoxifen inducible Cre transgene. PDK1 was then deleted from these CTL by tamoxifen treatment and the gene expression pattern determined by microarray. Tamoxifen treated PDK1wt/wt TamoxCre+ CTL generated from 3 PDK1wt/wt TamocCre+ mice were used as a control. In separate experiments CTL were were generated from 3 wild-type mice and then half the CTL generated from each mouse were treated with the Akt inhibitor AktI-1/2. The gene expression patterns of the AktI treated and the untreated CTL were then compared by microarray.
Project description:In cytotoxic T cells (CTL), Protein Kinase B /Akt is activated by the T cell antigen receptor (TCR) and the cytokine Interleukin 2 (IL2), in part by phosophorylation of Akt by Phospholipid dependent kinase 1 (PDK1). The role of PDK1 and Akt in CTL has however not been fully defined. In order to explore the relative roles of these kinases in CTL we used microarrays to profile the gene expression of control and PDK1 null CTL. In separate experiments we compared the gene expression profiles of control and Akt inhibitor treated CTL.
Project description:Comparison of transcriptional profile of CD8 cytotoxic T lymphocytes terated with the mTORC1 inhibitor rapamycin or the mTOR inhibitor KU-0063794 and comparison with proteomic analysis. Abstract: High resolution mass spectrometry maps the cytotoxic T lymphocyte (CTL) proteome and the impact of mammalian target of rapamycin complex 1 (mTORC1) on CTL. We show that the CTL proteome is dominated by metabolic regulators and granzymes and that mTORC1 selectively represses and promotes expression of a protein subset (~10%) including key CTL effector molecules and signaling proteins. mTORC1 also controlled flux through a subset of metabolic pathways rather than acting as an on/off switch for global CTL metabolism. Proteomic data highlighted the potential for mTORC1 negative control of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production in CTL. Further work revealed that mTORC1 represses PIP3 production and determines the mTORC2 requirement for activation of the serine/threonine kinase AKT. Unbiased proteomic analysis thus provides a comprehensive understanding of CTL identity and mTORC1 control of CTL function.