Project description:The present studies consist of five proteomic experiments: proteomic profiling treated by apigenin; DARTS coupled MS analysis to identify the potential directed proteins targets of apigenin; Identification of the potential directed protein targets of biotin-apigenin by pull-down coupled MS analysis; proteomic profiling of senescent cells upon treatment with MJ33; Identification of the proteins interacting with PRDX6 by Co-IP coupled MS analysis.
Project description:Illumina single-end sequencing of Hela_2 (HeLa cell line). While the number and identity of proteins expressed in a single human cell type is currently unknown, this fundamental question can be addressed by advanced mass spectrometry (MS)-based proteomics. On-line liquid chromatography coupled to high resolution MS and MS/MS yielded more than 150,000 unique peptides that identified more than 10,000 different human proteins encoded by more than 9,000 human genes. Deep transcriptome sequencing revealed transcripts for nearly all detected proteins. We show that the abundances of more than 90% of proteins and transcripts fall within a 10,000-fold range, and allocate the proteome to different compartments, complexes and functions. Comparisons of the proteome and the transcriptome, and analysis of protein complex databases and GO categories, suggest that we achieved almost complete coverage of the functional transcriptome and the proteome of a single cell type.
Project description:This study reports the interactome of HMGB1 in prostate and ovarian cancer cells, which includes a variety of proteins involved in diverse cellular processes. Subcellular fractionation is used to map the distribution of interacting proteins. In the nucleus, HMGB1 interacts directly with NuRD complex subunit RBBP7, as confirmed by yeast two-hybrid. Amongst HMGB1 interacting proteins are also subunits of the THOC complex and the septin complex. THOC5 IP-MS experiments confirmed that THOC proteins interact with septins and also with RAB11.
Project description:Interacting partners of TSG-6 were analyzed by coimmunoprecipitation (co-IP) coupled with liquid chromatography tandem mass spectrometry (LC–MS/MS)
Project description:Karyopherin beta 1 (Kpnβ1) is the principal nuclear importer of cargo proteins and plays a role in many cellular processes. Its expression is upregulated in cancer and essential for cancer cell viability, thus the identification of its binding partners might help in the discovery of anti-cancer therapeutic targets and cancer biomarkers. Herein, we applied immunoprecipitation coupled to mass spectrometry (IP- MS) to identify Kpnβ1 binding partners in normal and cancer cells. IP-MS identified 100 potential Kpnβ1 binding partners in non-cancer hTERT-RPE1, 179 in HeLa cervical cancer, 147 in WHCO5 oesophageal cancer and 176 in KYSE30 oesophageal cancer cells, including expected and novel interaction partners. 38 binding proteins were identified in all cell lines, with the majority involved in RNA metabolism. 18 binding proteins were unique to the cancer cells, with many involved in protein translation. Western blot analysis validated the interaction of known and novel binding partners with Kpnβ1 and revealed enriched interactions between Kpnβ1 and select proteins in cancer cells, including proteins involved in cancer development, such as Kpnα2, Ran, Crm1, CCAR1 and FUBP1. Together, this study shows that Kpnβ1 interacts with numerous proteins, and its enhanced interaction with certain proteins in cancer cells likely contributes to the cancer state.
Project description:p53-/- HCT116 cells transduced with the control empty vector or vectors expressing wtp53 or R175H mutp53 were employed for co-IP followed by LC-MS/MS analysis. The potential wtp53 or R175H mutp53-interacting proteins are listed.
Project description:The accompanying dataset is the result of a systematic study to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae. We immunopurified, via the use of integrated, C-terminal GFP and 9Myc tags, the five actomyosin motors, Myo1, Myo2, Myo3, Myo4, and Myo5; the kinesin-like proteins Kar3, Kip1, Kip2, Kip3, and Smy1; and the dynein, Dyn1, from S. cerevisiae. Cells were either treated with formaldehyde or with the small molecule latrunculin B. We used formaldehyde crosslinking to stabilize associations between motors proteins and interacting RNAs before IP. Yeast cells growing exponentially in rich medium were treated with formaldehyde, lysed and sonicated, then incubated with magnetic beads coupled to monoclonal antibodies against either 9Myc or GFP to isolate the tagged motor proteins along with any associated RNAs. After IP, the formaldehyde crosslinks were reversed and the enriched RNAs and RNAs purified from the corresponding total lysate were amplified and labeled respectively with Cy5 and Cy3, then jointly hybridized to custom-made, S. cerevisiae oligonucleotide microarrays (Hogan et al., PLoS Biology, 2008). Alternatively, the addition of a low concentration of latrunculin B (2 ug/ml) to live cells to partially solubilize the actin cytoskeleton allowed for successful IP of the motor proteins and associated mRNAs without the need for a chemical crosslinker. In both the case of latrunculin B and formaldehyde treatment, we also performed IPs in which the untagged parent yeast strains were processed for IP and microarray analysis in a manner identical to that of the tagged strains (labeled as mock). transcription profiling
Project description:Examine the potential interacting proteins of QQS by IP-MS analysis using Arabidopsis anthers of the QQS-OE and WT at flower stage 12-15.