The endosomal sorting adaptor HDPTP is required for ephrinB EphB signalling in cell collapse and motor axon guidance. Sci Rep, 2019
ABSTRACT: Spectral count files from EphB2 BioID experiment found in "The endosomal sorting adaptor HD-PTP is required for ephrin-B:EphB signalling in motor axon guidance.". The parameters and protocol are described in detail in the methods section. In brief, we over expressed BirA* C-terminal tagged EphB2 in HEK 293 cells. We stimulated these cells with either media (no ligand), Fc or ephrin-B2-Fc in order to capture the protein landscape of ephrin-B2:EphB2 forward signalling.
BirA*-Flag-EGFP controls are VL_20150825_COT_05_AK.raw, VL_20150825_COT_06_AK.raw, VL_20150825_COT_07_AK.raw, VL_20150825_COT_08_AK.raw
Empty Vector controls are VL_20151116_COT_01_HEK293_pcDNA5EV_AK.raw, VL_20151116_COT_02_HEK293_pcDNA5EV_AK.raw, VL_20151116_COT_03_HEK293_pcDNA5EV_AK.raw, VL_20151116_COT_04_HEK293_pcDNA5EV_AK.raw
EphB2_Fc-BirA*-Flag samples are VL_20160623_COT_01_AK.raw, VL_20160718_COT_01_AK.raw, VL_20161116_COT_05_AK.raw, VL_20161116_COT_06_2_AK.raw
EphB2_NoLigand-BirA*-Flag samples are VL_20160630_COT_01_AK.raw, VL_20160630_COT_02_AK.raw, VL_20161116_COT_03_AK.raw, VL_20161116_COT_04_AK.raw
EphB2_EB2-BirA*-Flag samples are VL_20160623_COT_02_AK.raw, VL_20160718_COT_02_AK.raw, VL_20161118_COT_01_AK.raw, VL_20161118_COT_02_AK.raw
Project description:Yeast enolase (Eno2p) conjugated with EGFP and Flag-tag (Eno2p-EGFP-FLAG) and Eno2p with V22A substitution (Eno2V22Ap) conjugated with EGFP and Flag-tag (Eno2V22Ap-EGFP-FLAG) were produced in baker's yeast S. cerevisiae. After semi-anaerobic culture at 30 ˚C for 12h, cells producing Eno2p-EGFP-FLAG formed fluorescent foci, while cells producing Eno2V22Ap-EGFP-FLAG did not. The cells were collected and lysed, and proteins Eno2p-EGFP-FLAG or Eno2V22Ap-EGFP-FLAG and the associated proteins were coimmunoprecipitated using ANTI-FLAG M2 affinity gel and analyzed. Data contain two biological replicates and two technical replicates (n = 4). As the results, 96 proteins were detected with both recombinant Eno2p-EGFP-FLAG and Eno2V22Ap-EGFP-FLAG protein, 29 proteins were detected only with recombinant Eno2p-EGFP-FLAG protein, and 16 proteins were detected only with recombinant Eno2V22Ap-EGFP-FLAG protein. Data Processing/Data Analysis: The separated analytes were detected on an LTQ Velos linear ion trap mass spectrometer (Thermo Scientific). For data-dependent acquisition, the method was set to automatically analyze the five most intense ions observed in the MS scan. The mass spectrometry data were used for protein identification by the Mascot search engine on Protein Discoverer software (ver. 1.2, Thermo Scientific) against the information in the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org). Search parameters for peptide identification included a precursor mass tolerance of 1.2 Da, a fragment mass tolerance of 0.8 Da, a minimum of one tryptic terminus, and a maximum of one internal trypsin cleavage site. Cysteine carbamidomethylation (+57.021 Da) and methionine oxidation (+15.995 Da) were set as a differential amino acid modification. The data were then filtered at a q value ≤ 0.01 corresponding to 1% FDR at the spectral level.
Project description:It has been demonstrated that Ipr1 participate in gene expression regulation during Mycobatrium infection. To better understand whether Ipr1 can regulate gene expression by interacting with gene promoter directly or not, we performed ChIP-seq to identify interaction between Ipr1 and DNA fragments. Overall design: Raw-Bap-Ipr1-P2A-BirA and Raw-Bap-P2A-BirA (negative control) cells were used for ChIP-seq, Flag (FlagChIP bond) and biotin (BioChIP bond) tags were used to isolate Ipr1-DNA complexes.
Project description:We studied the variations of mRNA amounts after Flag-EVI1, Flag-EVI1Δ324, or Flag expression in HeLa cells. Despites EVI1 discovery in 1988, its recognized role as a dominant oncogene in myeloid leukemia and more recently in epithelial cancers, only a few target genes were known and it was not clear why EVI1 was involved in cancer progression. Here we obtained the genomic binding occupancy and expression data for EVI1 in human cells. We identified numerous EVI1 target cancer genes and genes controlling cell migration and adhesion. Moreover, we characterized a transcriptional cooperation between AP1 and EVI1 that regulated proliferation and adhesion through a feed-forward loop. This study provides human genome-wide mapping and expression analyses for EVI1 that will be useful for the research community. 12 samples were collected. Each condition was done in 4 replicates, collected 24 hours after transfection (for mild expression of EVI1 or EVI1Δ324). Transfections with Flag-expressing vector were used as controls.
Project description:Identification of interacting partners of the Partner and Localizer of BRCA2 (PALB2), essential regulator of DNA repair by homologous recombination. Interacting partners of PALB2 were purified by GFP pull down from asynchronous HEK293 Flp-In T-REx cells, exogenously expressing Flag-EGFP tagged PALB2 at a similar level than the endogenous PALB2 level. Before GFP pull down, whole cell protein extracts were pre-cleared on IgG agarose beads to decrease binding of non-specific proteins during GFP pull down. GFP pull down were performed using GFP-Trap agarose beads (Chromotek) and washed at low salt concentration (150mM NaCl), to maintain interactions of low affinity binding protein partners. As a negative control, Flag-EGFP interacting proteins were purified, following the same protocol as described for the purification of Flag-EGFP PALB2 interacting proteins. Experiments were performed in triplicate.
Project description:Signalling by target-derived neurotrophins is essential for the correct development of the nervous system and its maintenance throughout life. Several aspects concerning the lifecycle of neurotrophins and their receptors, tropomyosin receptor kinases (Trks) and p75NTR, have been characterised over the years, including formation of activated ligand-receptor complexes, their endocytosis, trafficking and signalling. However, the molecular mechanisms directing the sorting of activated neurotrophin receptors to their final cellular destination are not completely understood. Previously, our laboratory identified Bicaudal-D1 (BICD1), a dynein motor adaptor, as a key factor for lysosomal degradation of BDNF-activated TrkB and p75NTR in motor neurons. Here, we deciphered the mechanism responsible for this sorting process. Using a proteomic approach, we identified protein tyrosine phosphatase, non-receptor type 23 (PTPN23), a member of the endosomal sorting complexes required for transport (ESCRT) machinery, in the BICD1 interactome. Molecular mapping revealed that PTPN23 is not a canonical BICD1 cargo; instead, PTPN23 binds the N-terminus of BICD1, which is also essential for the recruitment of cytoplasmic dynein. In line with the BICD1 knockdown phenotype, loss of PTPN23 leads to increased accumulation of BDNF-activated p75NTR and TrkB in swollen vacuole-like compartments, suggesting that neuronal PTPN23 is a novel regulator of the endocytic sorting of neurotrophin receptors.