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CMK LC-MS/MS after CoIP of ARID3A

ABSTRACT: In order to determine proteins interacting with ARID3A in the ML-DS cell line CMK, we performed CoIP of endogenous ARID3A followed by LC-MS/MS

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Jan-Henning Klusmann

LAB HEAD: Jan-Henning Klusmann

PROVIDER: PXD025027 | Pride | 2023-03-11

REPOSITORIES: Pride

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Publications

The megakaryocytic transcription factor ARID3A suppresses leukemia pathogenesis.

Alejo-Valle Oriol O Weigert Karoline K Bhayadia Raj R Ng Michelle M Issa Hasan H Beyer Christoph C Emmrich Stephan S Schuschel Konstantin K Ihling Christian C Sinz Andrea A Zimmermann Martin M Wickenhauser Claudia C Flasinski Marius M Regenyi Eniko E Labuhn Maurice M Reinhardt Dirk D Yaspo Marie-Laure ML Heckl Dirk D Klusmann Jan-Henning JH

Blood 20220201 5

Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndro ...[more]

PMID: 34570885

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RUNX1 isoforms in leukemogenesis

Project description:Aneuploidy and structural aberrations affecting chromosome 21 (Hsa21) are the most frequent in cytogentic events in acute myeloid leukemia. However, it remains unclear why leukemic blasts select for amplifications of Hsa21 or parts of it and why children with Down syndrome (i.e. trisomy 21) are at a high risk of developing leukemia. Here, we propose that disequilibrium of the RUNX1 isoforms and resultant RUNX1A dominance are key to trisomy 21-associated leukemogenesis. Using a Hsa21-focussed CRISPR-Cas9 screen, we uncovered a strong and specific RUNX1 dependency in myeloid leukemia associated with Down syndrome (ML-DS). High levels of RUNX1A – as seen in ML-DS – synergized with the pathognomonic Gata1s mutation in ML-DS pathogenesis, an effect that was reversed upon restoration of the normal RUNX1A:RUNX1C equilibrium. Mechanistically, RUNX1A displaces RUNX1C from its endogenous binding sites and recruits the MYC cofactor MAX to induce oncogenic programs and perturb normal differentiation. This presents a therapeutic vulnerability that can be exploited by interfering with MYC:MAX dimerization. Our study highlights the importance of alternative splicing in leukemogenesis, and paves the way for developing specific and targeted therapies for ML-DS as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.

2023-05-10 | PXD030616 | Pride

Identification of GltB-interacting proteins by CoIP-MS

Project description:To investigate whether GltB possess more function, Co-immunoprecipitation coupled with Mass Spectrometry (CoIP-MS) was used to isolate the putative GltB binding proteins.We used strains PAO1/p-gtrS-YFP and ΔgltB/p-gtrS-YFP to ensure the membrane protein GtrS of great interest is at high levels. A target GltB-specific antibody anti-GltB was used and ΔgltB/p-gtrS-YFP was set up as a negative control. The whole gel lane was divided into small fractions and in-gel digested for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Control sample was also analyzed in parallel to distinguish the background proteins.

2021-09-09 | PXD020213 | Pride

Chromatin immunoprecipitation of yeast and CoIP-chip of nucleosome-enriched chromatin after immunoprecipitation for specific factors

Project description:ChIP-chip & CoIP-chip of nucleosome enriched chromatin after IP of specific factors

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Identification of SMIM26 interacting proteins

Project description:The CoIP proteins were digested by FAST and peptides were used to analysis.

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CoIP of Nrl1 from normal and irradiated fission yeast cells

Project description:Nrl1-associated proteins were isolated from wild-type cells untreated or exposed to 100Gy of ionising radiation, and then subjected to LC-MSMS analysis. Identifications were carried out using trypsin as protease.

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Grass carp SERPINA1 CoIP with hepatopancreas mass spectrometry analysis

Project description:SERPINA1, a member of the serine protease inhibitor family, plays a role in viral infection and inflammation by regulating the activities of serine and cysteine proteases. To further investigate the antiviral role SERPINA1 played in GCRV (Grass carp Reovirus) infection, a polyclonal antibody of SERPINA1 was prepared, and the protein interacting with SERPINA1 was screened by CoIP/MS in grass carp hepatopancreas tissue. Samples of hepatopancreas tissues from grass carp (n=3) before (healthy) and 8 days after the infection (post-infection) were selected. Grass carp were infected by intraperitoneal injection. The total tissue proteins were extracted according to the manufacturer’s instructions for cell lysate (Beyotime, Shanghai, China). The types and abundance of proteins bound to SERPINA1 before and after the infection were detected and analyzed using CoIP-MS.

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miRNA profiling of Jurkat with stably integrated HTLV1-LTR in the presence of HTLV-1 Tax

Project description:RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4(+) T-cell line, Jurkat. Jurkat cells with stably integrated HTLV1-LTR were transfected with either pCMV-Tax or pCMV. Total miRNA was isolated, labeled and hybridized to the miRNA microarray as described by the manufacturer (Invitrogen). The hybridized slides were then analyzed on a GenePix scanner.

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