PXD048580-1
PXD048580 is an original dataset announced via ProteomeXchange.
Dataset Summary
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 |
Dataset History
Revision | Datetime | Status | ChangeLog Entry |
---|---|---|---|
0 | 2024-01-15 21:04:21 | ID requested | |
⏵ 1 | 2024-02-17 17:43:58 | announced |
Publication List
Dataset with its publication pending |
Keyword List
submitter keyword: MCPH1 complex |
Contact List
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 |
Full Dataset Link List
iProX dataset URI |