Project description:Pull-down of poly(A)-mRNA cross linked proteins using two cross-linking methods (conventional cross-linking and PAR-cross-linking) to identify all mRNA-binding proteins (GO:0003729). The provided data is quantitative proteomic data for comparison of cross-linking and control samples.
Project description:To gain an insight into the PRE DNA-binding protein regulatory network, here, using immunoaffinity purification coupled to the high throughput mass spectrometry, we isolated factors associated with the Combgap, Psq, Zeste and Adf1 PRE DNA-binding proteins. We show that Combgap and Zeste are more tightly associated with the Polycomb repressive complex 1 (PRC1), while Psq interacts strongly with the TrxG proteins, including the BAP SWI/SNF complex. The Adf1 interactome contained Mediator subunits as the top interactors. In addition, Combgap efficiently interacted with AGO2, NELF, and TFIID. Combgap, Psq, and Adf1 have architectural proteins in their networks. We further investigated the existence of direct interactions between different PRE DNA-binding proteins and demonstrated that Combgap-Adf1, Psq-Dsp1, and Pho-Spps can interact in the yeast two-hybrid assay. Overall, our data suggest that Combgap, Psq, Zeste and Adf1 are associated with the protein complexes implicated in different regulatory activities and indicate their potential multifunctional role in the regulation of transcription.
Project description:In this project, we identified a novel RNA-binding protein, MHZ9. And we analyzed the potential proteins interacted with MHZ9 through immunoprecipitation-mass spectrometry (IP-MS). The N-terminal domain of MHZ9 (MHZ9-N) contains a putative RNA splicing and modification domain PRP4. To identify RNA binding sites in the MHZ9-N. We performed XRNAX-IP-MS assay.
Project description:We sought to determine whether the vault complex component Poly (ADP-ribose) Polymerase 4 (PARP4) would impact the identity or relative enrichment of proteins that interacted with the vault. We conducted co-immunoprecipitation (co-IP) experiments targeting the human major vault protein (MVP) using a synthetic antigen binding reagent (sAB) generated for this study. Experimental co-IP's were conducted by adding the anti-MVP sAB (called VM1) to WT A549 cells and to cells from which PARP4 had been depleted using CRISPR/Cas9. To control for background, we concurrently processed samples to which we had added an anti-GFP sAB (called H6) that was highly similar to our anti-MVP reagent but was not specific for any human proteins. PARP4, as well as a number of other proteins, were significantly enriched in the experimental co-IP from the WT versus PARP4 KO cells. Overall, we identified 34 hits that were either unique to or differentially enriched in the WT cell experimental co-IP, 16 hits that were unique to the PARP4 KO experimental co-IP, and 18 "shared" hits that were present in each experimental co-IP dataset without significant differential enrichment in either.
Project description:Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.
Project description:Proteins that may interact with HERD-1 were identified with Flag IP-mass in animals, which were tagged with 3xflag::GFP before the stat condon of HERD-1 in C.elegans.
Project description:Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway. For microarray screening, Invitrogen Protoarray v4.0 protein microarrays were used. Human NEMO expressed as a C-terminal GST fusion was purified from E. coli lysates and labelled with biotin. NEMO or biotinylated GST were applied to the microarrays and binding partners detected using streptavidin-Alexa Fluor 647. Significant interactors on both arrays were detected using Invitrogen Protoarray Prospector software and a Z-score cutoff of 3.0. Following subtraction of interactors present on the GST control array, a final set of significant NEMO interactors was derived. Full experimental details are supplied in Fenner, B. J., Scannell, M. & Prehn, J. H. M. (2010). Expanding the substantial interactome of NEMO using protein microarrays. PLoS ONE (in press).
Project description:Vacuoles and lysosomes are single-membrane-bound organelles involved in diverse functions such as intracellular digestion and storage or secretion of metabolites. To understand their origin, evolution and fundamental features, the identification of proteins comprising these compartments in missing links would be invaluable. So, we isolated the vacuoles from Cyanidioschyzon merolae, which is considered to be one of the most primitive photosynthetic eukaryotes, and identified 46 proteins by matrix-assisted laser desorption/ionization time of flight-mass spectrometry. Keywords: peptide mass fingerprinting, MALDI-TOF
Project description:To explore the mechanism by which the S100A11 protein activate macrophage, we screened potential proteins that interacted with S100A11 using immunoprecipitation-mass spectrometry (IP-MS) analysis. IP was performed in 293T cells overexpressing FLAG-tagged Vector or S100A11