Dataset Information

50_100uM_DSBU_XL-MS_PHB2(V1)-Myc_HEK293

ABSTRACT: 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.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD011941 | JPOST Repository | Thu Sep 05 00:00:00 BST 2019

REPOSITORIES: jPOST

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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

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Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

			Action	DRS
	Lumos_100uM_CIDETDFT.raw	Raw
	Lumos_100uM_CIDETDIT.raw	Raw
	Lumos_100uM_HCD.raw	Raw
	Lumos_50uM_CIDETDFT.raw	Raw
	Lumos_50uM_CIDETDIT.raw	Raw