PXD038215

PXD038215 is an original dataset announced via ProteomeXchange.

Title	Potential interactors of NLRP3 in NLRP3-inducible THP-1 cells treated with LPS
Description	The NLRP3-inducible THP-1-cell line was generated by introducing Flag-WT NLRP3 into NLRP3 KO THP-1 cells through a tetracycline (Tet) responsive element (TRE) promoter (Tet-On) lentiviral infection system. Cells were treated with doxycycline (Dox, 200 ng/mL, Sigma-Aldrich, cat# D9891) for 18 h to induce the expression of Flag-NLRP3 in THP-1-derived macrophages, followed by treatment with lipopolysaccharide (LPS, 200 ng/ml, Sigma-Aldrich, cat# L4524) for 3 h. We used the cells without doxycycline treatment as a negative control and mixed the cells from three individual flasks for mass spectrometry. The cells were collected in low-salt lysis buffer (50 mM Tris-HCl (GCRF, China), pH 8.0, 150 mM NaCl (GCRF, China), 1 mM EDTA (Vetec), 1 mM DTT (Sigma-Aldrich) and 0.5% IGEPAL CA630 (Sigma-Aldrich) containing phosphatase inhibitor (Roche) and EDTA-free protease inhibitor (Biosharp)) and incubated on a rocker with ice for 30 min and centrifuged at 12000 g/4 °C for 15 min to obtain cell lysates. The cell lysates were immunoprecipitated with an anti-Flag antibody. The beads were washed three times with low-salt lysis buffer, separated by SDS-PAGE, and then stained with Coomassie Blue. The entire lane was excised and digested with trypsin at 37 °C for 20 h. The enzymatic hydrolysis product was desalted, lyophilized and redissolved in 0.1% FA solution for chromatography-tandem mass spectrometry (LC-MS/MS) identification of peptide mixtures at Applied Protein Technology (aptbiotech, Inc. Shanghai, China) with a Q Exactive mass spectrometer (Thermo Fisher, California, USA). RAW files generated from the spectrometer were subjected to Proteome Discoverer 1.4 software for protein identification.
HostingRepository	iProX
AnnounceDate	2022-11-18
AnnouncementXML	Submission_2022-11-18_03:49:31.571.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Liqiu Wang
SpeciesList	scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2022-11-18 03:49:19	ID requested
⏵ 1	2022-11-18 03:49:31	announced

Dataset with its publication pending

submitter keyword: NLRP3, mass spectrometry, proteomics

Jun Cui
contact affiliation	Sun Sun Yat-sen University
contact email	cuij5@mail.sysu.edu.cn
lab head
Liqiu Wang
contact affiliation	Sun Yat-sen University
contact email	1812604613@qq.com
dataset submitter

iProX dataset URI