Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

BioID2_BirA-MAVS_CLPB-BirA_PHB1-BirA_HEK293

ABSTRACT: HEK293 cells that stably express MAVS, CLPB or PHB1 fused at C-terminus with the biotin ligase from A. aeolicus were incubated with 100 μM biotin for 18 h. The mitochondrial fractions were lysed in RIPA buffer, and the clarified supernatants were incubated with streptavidin agarose beads for 2 h at 4° C. Proteins on the beads were digested by adding trypsin/Lys-C mix for 16 h at 37°C and analyzed by LC-MS/MS. Label-free quantification was performed using Proteome Discoverer 2.2 software.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD014218 | JPOST Repository | Thu Sep 05 00:00:00 BST 2019

REPOSITORIES: jPOST

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	BioID2_CLPB.raw	Raw
	BioID2_Control.raw	Raw
	BioID2_MAVS.raw	Raw
	BioID2_PHB1.raw	Raw
	MAVS_CLPB_PHB1_BioID2.xlsx	Xlsx

Items per page:

1 - 5 of 5

Publications

Structural Basis of Mitochondrial Scaffolds by Prohibitin Complexes: Insight into a Role of the Coiled-Coil Region.

Yoshinaka Takahiro T Kosako Hidetaka H Yoshizumi Takuma T Furukawa Ryo R Hirano Yu Y Kuge Osamu O Tamada Taro T Koshiba Takumi T

iScience 20190903

The coiled-coil motif mediates subunit oligomerization and scaffolding and underlies several fundamental biologic processes. Prohibitins (PHBs), mitochondrial inner membrane proteins involved in mitochondrial homeostasis and signal transduction, are predicted to have a coiled-coil motif, but their structural features are poorly understood. Here we solved the crystal structure of the heptad repeat (HR) region of PHB2 at 1.7-Å resolution, showing that it assembles into a dimeric, antiparallel coil ...[more]

PMID: 31522117

Similar Datasets

BioID2_PHB2(V1)-(WT_KKK_I225P)

Project description:HEK293 cells that stably express wild-type (WT), E229/231/233K (KKK), or I225P mutant PHB2(V1) fused at C-terminus with the biotin ligase from A. aeolicus were incubated with 100 μM biotin for 18 h. The mitochondrial fractions were lysed in RIPA buffer, and the clarified supernatants were incubated with streptavidin-agarose beads for 2 h at 4° C. Proteins on the beads were digested by adding trypsin/Lys-C mix for 16 h at 37° C and analyzed by LC-MS/MS. Label-free quantification was performed using Proteome Discoverer 2.2 software.

2019-09-05 | PXD011946 | JPOST Repository

BioID2_PHB2(V1)_PHB2(V3)-BirA-HA_HEK293

Project description:HEK293 cells that stably express PHB2 (V1 or V3) fused at C-terminus with the biotin ligase from A. aeolicus were incubated with 100 μM biotin for 18 h. The mitochondrial fractions were lysed in RIPA buffer, and the clarified supernatants were incubated with streptavidin agarose beads for 2 h at 4° C. Proteins on the beads were digested by adding trypsin/Lys-C mix for 16 h at 37° C. Three biological replicates of the samples were individually prepared and analyzed by LC-MS/MS. Label-free quantification was performed using Proteome Discoverer 2.2 software.

2019-09-05 | PXD011939 | JPOST Repository

Mapping of Notch1 binding sites in a mouse immature T cell line

Project description:We used an immature mouse T cell line engineered to express a biotinylated form of the cleaved form of Notch1 (ICN1). ICN1-bound sites were precipitated with streptavidin-coated beads and subjected to ChIP-sequencing. Beko cells correspond to a spontaneous T lymphoma immature cell line derived from a TCRb deficient mouse. These cells were engineered to express a biotin-tagged-ICN1 and the bacterial biotin ligase BirA (Bio-ICN1) or just BirA as a control (Bio). Chromatin from both cell lines was subjected to strepatavidin-mediated precipitation and subjected to sequencing with the Illumina GAII sequencer as single end 36 base pair reads.

2014-04-02 | E-GEOD-46114 | biostudies-arrayexpress

Effects of CEBPA isoform expression on NB4 cells

Project description:Here we investigated the effects of CEBPA transcription factor expression on myeloid NB4 cells. The sequence of rat CEBPA was C-terminally fused to a promiscuous biotin ligase tag (BirA*) and NB4 cell lines were engineered to express the fusion protein under the control of a doxycycline inducible promoter. Three different NB4 cell lines were investigated that expressed (i) BirA* tag alone (ii) full length CEBPA isoform (P42) fused to BirA* (iii) truncated CEBPA isoform (P30) fused to BirA*. Cells were seeded in media supplemented with or without doxycycline.

2021-06-26 | E-MTAB-9947 | biostudies-arrayexpress

A native chromatin purification system for epigenomic profiling in C. elegans

2009-12-08 | GSE18898 | GEO

A native chromatin purification system for epigenomic profiling in C. elegans

Project description:We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf All experiments were done using two channels per chip, comparing DNAs extracted from either salt-extracted or insoluble chromatin to whole nuclear chromatin, whole nuclear chromatin to randomly fragmented genomic DNA, streptavidin-bound biotin-tagged histone-variant-containing chromatin to salt-extracted chromatin, gel-purified mononucleosomes to whole EDTA-extracted soluble chromatin, or streptavidin-bound biotin-tagged histone-variant-containing chromatin to whole EDTA-extracted soluble chromatin to randomly fragmented DNA from embryo nuceli.

2010-05-19 | E-GEOD-18898 | biostudies-arrayexpress

100uM_DSBU_XL-MS_PHB1-Myc_HEK293

Project description:Mitochondria were isolated from HEK293 cells that stably express PHB1 with an C-terminal 3×Myc tag, and were resuspended in a cross-linking buffer [1 × PBS (pH 7.2) and 100 μM disuccinimidyl didutyric urea (DSBU) for 30 min at room temperature. The cross-linked mitochondria were then lysed in RIPA buffer, and the clarified supernatants were incubated with anti c-Myc agarose beads. Proteins on the beads were digested by adding trypsin/Lys-C mix for 16 h at 37°C. The resultant peptides were subjected to LC-MS/MS analysis using a Q Exactive Plus spectrometer. Raw data were analyzed using Proteome Discoverer 2.2 with the XlinkX node to identify cross-linked peptides.

2019-09-05 | PXD014217 | JPOST Repository

Off the shelf proximity biotinylation using ProtA-TurboID

Project description:Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or CRISPR-based tagging, thus prohibiting such assays in (primary) cell types that are difficult to transfect. We recently developed an ‘off the shelf’ proximity biotinylation method, which makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo-antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. Here, we present a detailed protocol for this method

2022-10-17 | PXD030963 | Pride

50_100uM_DSBU_XL-MS_PHB2(V1)-Myc_HEK293

Project description:Mitochondria were isolated from HEK293 cells that stably express PHB2(V1) with an C-terminal 3×Myc tag, and were resuspended in a cross-linking buffer [1 × PBS (pH 7.2) and 50 or 100 μM disuccinimidyl didutyric urea (DSBU) for 30 min at room temperature. The cross-linked mitochondria were then lysed in RIPA buffer, and the clarified supernatants were incubated with anti c-Myc agarose beads. Proteins on the beads were digested by adding trypsin/Lys-C mix for 16 h at 37°C. The resultant peptides were divided into two halves and subjected to LC-MS/MS analysis using both Q Exactive Plus and Orbitrap Fusion Lumos spectrometers. Raw data were analyzed using Proteome Discoverer 2.2 with the XlinkX node to identify cross-linked peptides.

2019-09-05 | PXD011941 | JPOST Repository

Isolation and determination of PI(4,5)P2 proximal proteins

Project description:We screened proteins in close proximity to PI(4,5)P2 in epithelial cells. We established HaCaT cells stably expressing APEX2 N-terminally fused to the PH domain of PLCd1, which specifically binds to PI(4,5)P2. In the presence of hydrogen peroxide and biotin-phenol, proteins proximal to APEX2 are biotinylated by APEX2, allowing for their enrichment using streptavidin beads. APEX2-fused non-PI(4,5) P2-binding mutant of the PH domain of PLCd1 (R40L) was used as the negative control.

2022-03-26 | PXD031266 | Pride