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Proteins that interact with PHAX and UAP56 from HeLa nuclear extracts

ABSTRACT: HeLa nuclear extracts and RNase A were incubated with GST-PHAX and FLAG-UAP56 in the presence of ATP. The mixture was subjected to immunoprecipitation. The bound proteins were eluted with FLAG peptide. The eluate was subjected to GST pull-down. The bound proteins were treated with HRV 3C protease. Supernatants were analysed by SDS-PAGE and mass spectrometry.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ichiro Taniguchi

PROVIDER: PXD053480 | JPOST Repository | Sat Jun 28 00:00:00 BST 2025

REPOSITORIES: jPOST

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			Action	DRS
	Peptide%20Summary%20Report%20-a.pdf	Pdf
	Peptide%20Summary%20Report%20-b.pdf	Pdf
	Peptide%20Summary%20Report%20-c.pdf	Pdf
	Peptide%20Summary%20Report%20-d.pdf	Pdf
	Peptide%20Summary%20Report%20-e.pdf	Pdf

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Publications

The RNA helicase DDX39 contributes to the nuclear export of spliceosomal U snRNA by loading of PHAX onto RNA.

Taniguchi Ichiro I Hirose Tetsuro T Ohno Mutsuhito M

Nucleic acids research 20240901 17

RNA helicases are involved in RNA metabolism in an ATP-dependent manner. Although many RNA helicases unwind the RNA structure and/or remove proteins from the RNA, some can load their interacting proteins onto RNAs. Here, we developed an in vitro strategy to identify the ATP-dependent factors involved in spliceosomal uridine-rich small nuclear RNA (U snRNA) export. We identified the RNA helicase UAP56/DDX39B, a component of the mRNA export complex named the transcription-export (TREX) complex, an ...[more]

PMID: 39011894

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Identification of SOX2-bound mRNA

Project description:In vitro pull down assay was performed using recombinant GST and GST fusion SOX2 protein to precipitate total RNA from urothelial carcinoma cell line BFTC905 Unbound RNA from both reactions and matrix-associated RNA from SOX2 pull down reaction was extracted using TRIZOL reagent This experiment is to identify mRNA bound by SOX2 under in vitro experimental condition

2011-10-25 | E-GEOD-33207 | biostudies-arrayexpress

Genome-wide mapping of the TET3 CXXC domain binding sites

Project description:The TET3 CXXC domain has unique DNA binding properties. It binds to DNA in a cytosine-dependent manner that prefers binding to CpG dinucleotides but is not restricted by the CpG-content, distinct from other well-characterized CXXC domains. To map the TET3 CXXC domain binding sites across the human genome, we purified the GST-tagged TET3 CXXC domain protein and performed the GST pull-down assay using the genomic DNA purified from HEK293T cells. The enriched DNA fragments were then sequenced and aligned to human genome(hg19). We used the GST pull-down assay followed by DNA deep sequencing to map the DNA bound by the TET3 CXXC domain in vitro.

2012-12-10 | E-GEOD-41551 | biostudies-arrayexpress

Identification of SOX2-bound mRNA

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Integrative Genomics Positions MKRN1 as a Novel Ribonucleoprotein within the Embryonic Stem Cell Gene Regulatory Network

Project description:For identification of proteins that associate with Makorin1 (MKRN1) in RNA-dependent and RNA-independent manners, we affinity purified FLAG-tagged Makorin1 (MKRN1) from mouse embryonic stem cells constitutively expressing FLAG:MKRN1. Anti-FLAG control immunoprecipitations were performed from a FLAG vectrol control (FLAG:Ctrl) mouse embryonic stem cell line that did not express FLAG:MKRN1. Following FLAG immunoprecipitation, anti-FLAG beads from FLAG:MKRN1 and FLAG:Ctrl immunoprecipitations were split into separate tubes such that half of the beads were digested with 200Ã∞â≈ Ã≠Âµg/mL RNase A while the other half of the beads were undigested. RNase A-digested and undigested immunoprecipitates were subjected to LC-MS/MS analysis. Of the 48 RNA-related proteins previously identified to associate with FLAG:MKRN1, L1TD1, PABPC1, PABPC4, YBX1, IGF2BP1 and UPF1 were found to remain associated with FLAG:MKRN1 in the presence of RNase A.

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Effect of Drosophila immune proteins on gene expression in E. coli

Project description:Interaction between the host and invading pathogen determines the fate of both organisms during the infectious state. The host is equipped with a battery of immune reactions, while the pathogen displays a variety of mechanisms to compromise host immunity. Although bacteria alter their pattern of gene expression when they enter host organisms, studies to elucidate the mechanism behind this are only in their infancy. In the present study, we examined the possibility that host immune proteins directly participate in the change of gene expression in bacteria. To this end, Escherichia coli was treated with a mixture of the extracellular region of membrane-bound peptidoglycan recognition protein LC (PGRP-LC) and the antimicrobial peptide attacin of Drosophila, and subsequently subjected to DNA microarray analysis for the repertoire of mRNA. We identified nearly 200 genes whose mRNA increased after the treatment, and at least four of them were induced in response to PGRP-LC. One such gene, lipoprotein-encoding nlpI, showed a transient increase of its mRNA level in adult flies depending on PGRP-LC, and NlpI-lacking E. coli had a smaller pathogenic effect with lowered growth/viability than the parental strain in adult flies. These results suggest that a host immune receptor triggers a change of gene expression in bacteria simultaneously to their recognition of the invader and induction of immune responses. The E. coli strain BW25113 (2 x 10^9) suspended with insect saline (0.13 M NaCl, 4.7 mM KCl, 1.9 mM CaCl2), was incubated with a mixture of GST-attacin (0.125 Î¼M), GST-PGRP-LCa (0.5 Î¼M), GST-PGRP-LCx (1 Î¼M), and GST-PGRP-LCy (0.5 Î¼M) for 10 min at room temperature. As a negative control, incubation of E. coli was done in the presence of GST alone (3 Î¼M).

2015-01-13 | E-GEOD-61604 | biostudies-arrayexpress

RNA pull down assay of GST-ZFP36L1 (wildtype and mutant)

Project description:RNA pull down assay of GST-ZFP36L1 (wildtype and mutant)

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Transcriptome-wide mapping of MARF1-bound mRNAs

Project description:To comprehensively identify MARF1-associated mRNAs, we performed iCLIP using engineered HEK293 cells stably expressing a doxycycline (Dox)-inducible FLAG-tagged MARF1 that lacks RNAse activity. Our iCLIP analysis of MARF1 in HEK293 cells identified 108 mRNAs bound by MARF1 and demonstrates that MARF1 binds to the majority of these mRNAs via their 3’UTRs.

2020-06-16 | GSE149820 | GEO

GNPS - PAP-gamma associates with PAXT nuclear exosome to control the stability of PROMPT ncRNAs

Project description:Endogenous ZFC3H1 was tagged in 293T cells. Dignam cellular extracts where subjected to sequential FLAG and HA immunoprecipitation and elution. Complexes were then analyzed using mass spectrometry at the taplin mass spectrometry facility. The 2 samples are FLA-HA IP in wild-type 293T cells and Flag-HA IP in FH-ZFC3H1 293T cells.

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Interactome analysis of human Rab11a with proteins from influenza A virus infected cell lysates

Project 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.

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