PXD030058

PXD030058 is an original dataset announced via ProteomeXchange.

Title	Interactome analysis of human Rab11a with proteins from influenza A virus infected cell lysates
Description	The aim of this project was to determine which viral and host proteins bind to purified recombinant human GTPase Rab11a from lysates of HEK-293T cells infected with influenza A/WSN/33 (H1N1) virus. To this aim we used a GST pull-down assay. Approximately 8∙106 HEK-293T cells were infected with influenza A/WSN/33 (H1N1) virus at a multiplicity of infection of 5 (MOI=5) or mock infected. Twelve hours (h) post-infection cells were harvested by centrifugation at 1,000 rpm for 5 min and lysed for 1 h in lysis buffer [50 mM HEPES-NaOH pH 7.5, 200 mM NaCl, 0.5 % (v/v) IGEPAL CA-630 (Sigma-Aldrich), 1 mM β-mercaptoethanol, 1x PMSF, 1x protease inhibitor (Roche, complete mini, EDTA-free)] at 4 °C. Supernatants were collected by centrifugation at 13,000 rpm for 5 min at 4 °C. Approximately 0.2 mg of purified GST-tagged Rab11CA (constitutively active Rab11a with a Q70L substitution) or GST tag was bound to pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) (100 µl slurry per 1 ml sample volume) at 4 °C for 3 h in binding buffer [50 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 10 % (v/v) glycerol, 8 mM MgCl2, 10 µM GTP-γ-S (Abcam)]. GST-Rab11CA or GST tag bound beads were incubated with virus infected or mock infected cell lysates for 1 h at 4 °C followed by five washes in binding buffer. Rab11CA and bound proteins were released in the same buffer supplemented with 1 mM DTT and 0.1 mg human rhinovirus (HRV) 3C protease to cleave off Rab11CA from the GST tag. After 1 h incubation at 4 °C released proteins were cleared from the beads by centrifugation at 1,000 rpm for 5 min at 4 °C. GST pull-down protein fractions were used for analysis by mass spectrometry.
HostingRepository	PRIDE
AnnounceDate	2022-01-17
AnnouncementXML	Submission_2022-01-17_11:49:45.268.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Marjorie Fournier
SpeciesList	scientific name: Influenza A virus (A/WSN/1933(H1N1)); NCBI TaxID: 382835; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2021-11-30 07:50:16	ID requested
⏵ 1	2022-01-17 11:49:45	announced

Dataset with its publication pending

submitter keyword: Interactome, human Rab11a, influenza virus infection

Ervin Fodor
contact affiliation	Sir William Dunn School of Pathology, University of Oxford
contact email	ervin.fodor@path.ox.ac.uk
lab head
Marjorie Fournier
contact affiliation	University of Oxford
contact email	marjorie.fournier@bioch.ox.ac.uk
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/01/PXD030058

PRIDE project URI

• PRIDE
  1. PXD030058
     1. Label: PRIDE project
     2. Name: Interactome analysis of human Rab11a with proteins from influenza A virus infected cell lysates