PXD048580

PXD048580 is an original dataset announced via ProteomeXchange.

Title	Affinity purification of MCPH1protein complexes
Description	In order to identify the molecular mechanism of MCPH1 antagonizing necroptosis， we performed affinity purification of MCPH1 protein complexes in TSZ-treated L929 cells, L929 cells were stably expressing Flag-tagged MCPH1 and treated with T+S+Z (T: 50 ng/ml TNFα, S: 100 nM SmacM, Z: 20 μM zVAD) for 1 hour. The resulting cells were lysed in NETN buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40, containing protease and phosphatase inhibitors cocktail) on ice for 30 min. Cell debris were eliminated by centrifugation and the supernatant was incubated with anti-Flag beads. After the 4 hours incubation, beads were washed with 1 mL NTEN buffer for three times, beads were boiled in 30 μL 2×SDS loading buffer and the immunocomplexes were eluted. Samples were resolved on 10% SDS-PAGE and analyzed by mass spectrometry. To elucidate the molecular mechanism behind MCPH1 nuclear translocation upon DNA damage, we performed affinity purification of MCPH1 protein complexes in response to camptothecin (CPT) treatment. HEK293T cells stably expressing SFB-tagged MCPH1 were lysed in NETN buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40, containing protease and phosphatase inhibitors cocktail) on ice for 30 min. Cell debris were eliminated by centrifugation and the supernatant were obtained and incubated with 50 μL S-protein beads at 4 ℃. After the 4 hours incubation, beads were washed with 1 mL NTEN buffer for three times, beads were boiled in 30 μL 2×SDS loading buffer and the immunocomplexes were eluted. Samples were resolved on 10% SDS-PAGE and analyzed by mass spectrometry.
HostingRepository	iProX
AnnounceDate	2024-01-13
AnnouncementXML	Submission_2024-02-17_17:43:58.004.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hanqing He
SpeciesList	scientific name: Mus musculus; NCBI TaxID: 10090; scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive HF-X

Revision	Datetime	Status	ChangeLog Entry
0	2024-01-15 21:04:21	ID requested
⏵ 1	2024-02-17 17:43:58	announced

Dataset with its publication pending

submitter keyword: MCPH1 complex

Jianwei Wang
contact affiliation	Tsinghua University
contact email	wangjianwei@ihcams.ac.cn
lab head
Hanqing He
contact affiliation	Tsinghua University
contact email	hhq18@tsinghua.org.cn
dataset submitter

iProX dataset URI