Project description:Endogenous ZFC3H1 was tagged in 293T cells. Dignam cellular extracts where subjected to sequential FLAG and HA immunoprecipitation and elution. Complexes were then analyzed using mass spectrometry at the taplin mass spectrometry facility. The 2 samples are FLA-HA IP in wild-type 293T cells and Flag-HA IP in FH-ZFC3H1 293T cells.
Project description:Evaluation of binding partners of wild-type endogenously tagged flag-HA-dSetDB1 versus catalytic dead endogenously tagged flag-HA-250 dSetDB1 in Drosophila melanogaster early embryos
Project description:Cyclin D1 belongs to the core cell cycle machinery1, and it is frequently overexpressed in human cancers2. The full repertoire of cyclin D1 functions in normal development and in cancer cells is currently unknown. To address this question, here we introduce a novel approach that allows one to determine the set of cyclin D1-interacting proteins (D1 âinteractomeâ) and cyclin D1-bound genomic fragments (D1 âcistromeâ) in essentially any mouse organ, at any point of development or at any stage of cancer progression. Using this approach, we detected several novel tissue-specific interactors of cyclin D1. A significant number of these partners represent proteins involved in transcription. We show, using genome-wide location analysis3, that cyclin D1 occupies promoters of a very large number of genes in the developing mouse, where it binds in close proximity to transcription start sites. Bioinformatics analyses of cyclin D1-bound genomic segments in the developing embryo revealed DNA recognition sequences for several transcription factors. By querying SAGE libraries4, promoter CpG content5 and gene expression profiles of cyclin D1-null organs, we demonstrate that cyclin D1 binds promoters of highly expressed genes, and that it functions to activate or to repress gene expression in vivo. Analyses of cyclin D1 transcriptional targets reveal that cyclin D1 contributes to cell proliferation by upregulating genes required for S-phase entry and progression. Hence, cyclin D1 plays a broad transcriptional regulatory function in vivo during normal mouse development. We hypothesized that cyclin D1 may play role in transcription by interacting with transcriptional machinery at the promoters of target genes. To test this possibility, we employed chromatin immunoprecipitation coupled to DNA microarray analysis (genome-wide location analysis or ChIP-chip) to study association of cyclin D1 with genomic DNA sequences. In this procedure, a protein of interest is crosslinked to DNA, immunoprecipitated, and the bound DNA is hybridized to an array containing probes that span the genome. Since anti-FLAG antibodies have been successfully used for ChIP-chip in several different systems including murine cells, knock-in mice expressing tagged cyclin D1 provided us with a novel tool to query the association of cyclin D1 with the genome in vivo, in a living mouse.
Project description:To investigate the chromatin binding patterns of wildtype SOX10 versus schwannoma-derived mutant isoforms, we established SVG-p12 cell lines stably expressing either FLAG epitope tagged wildtype SOX10 or two different mutant isoforms of SOX10. We then performed chromatin immunoprecipitation-DNA sequencing on these SVG-p12 cell lines stably transduced with wildtype or mutant SOX10 isoforms using an anti-FLAG epitope antibody.
Project description:To explore the mechanism of pachytene pirna biogenesis, we employed a proteomics approach using flag-tagged adad2 transgenic mice expressed fully functional N-terminal epitope-tagged HA-ADAD2 and performed anti-FLAG immunoprecipitation coupled with quantitative mass spectrometry (IP-MS) from testis extracts of adult wt and transgenic mice.
Project description:To identify candidate mTOR-interacting molecules, we performed an anti-Flag immunoprecipitation (IP) followed by mass spectrometry (MS)-based proteomic analysis in 293T cells overexpressing Flag-tagged mTOR. We identified P4HA2 binding proteins with anti-HA IP followed by (MS)-based proteomic analysis in 293T cells overexpressing HA-tagged P4HA2. To speculate that mTOR can be hydroxylated by P4HA2. We used 293T cells overexpressing Flag-mTOR, Flag-mTOR/HA-P4HA2, and Flag-mTOR/HA-P4HA2-overexpressing cells treated with EDHB, we conducted an anti-Flag IP to capture and enrich mTOR protein, and the immunoprecipitates were subjected to MS analysis.
Project description:NPAC ChIP were performed by anti-Flag and anti-HA tandem affinity purification from HeLa stably expressing Flag-HA tagged NPAC from pOZ-N vector, and enrichement on chromosome 3, 21, and 22 were determined by chip microarray analysis using Affymatrix HumanTiling 2.0 arrays
Project description:Co-IP assays and Proteomics were performed to identify potential E3 ligases interacted with both cyclin D1 T286A and cyclin D1 T288A.
