Identification of proteins interacting with HA-FLAG-Eya1
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Georgia Dolios
LAB HEAD: Pin-Xian Xu
PROVIDER: PXD033955 | Pride | 2022-10-12
REPOSITORIES: Pride
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Project description:Arabidopsis ecotype Col-0 expressing RPL18 fused to His & Flag epitopes (RPL18-HF) under 35S control was used to discern the effects of Turnip mosaic virus (TuMV) infection on host translation initiation. Plants were grown in LC-1 soil in 12 hour days and infected by TuMV-GFP or mock-inoculated just prior to sending up bolts. Samples were taken from rosette leaves 10 days after inoculation. Only tissue fluorescing GFP was selected from the virus-infected samples. Similar tissue was sampled from mock-infected leaves. FLAG antibody was used to isolate RPL18-HF. The RNA co-immunoprecipitated with RPL18-HF is fully translation-initiated. This translation-initiated RNA, also referred to as polysomal RNA, was isolated and compared to total RNA under both mock and TuMV-infected states to find TuMV-induced changes in host translation initiation. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Jackson Moeller. The equivalent experiment is AT42 at PLEXdb.]
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Project description:The aim of this analysis is to identify the difference between interaction partners of MRG15 wild type and Chromodomain and MRG domain mutants upon UV irradiation. Therefore, MRG15 wild type and mutants (N-terminal FLAG tag) were overexpressed in U2OS cells. After UV irradiation, immunoprecipitation of the constructs was performed using FLAG M2 affinity gel. The constructs were eluted from the beads with 3xFLAG peptide.
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Project description:Here we examine key regulatory pathways underlying the transition from compensated hypertrophy (HYP) to decompensated heart failure (HF) and sudden cardiac death (SCD) in a guinea pig model by integrated multi-ome analysis. Relative protein abundances from sham-operated, HYP and HF hearts were assessed by iTRAQ shotgun LC-MS/MS. Metabolites were quantified by LC-MS/MS or GC-MS. Transcriptome profiles were obtained using DNA microarrays. The guinea pig HF proteome exhibited classic biosignatures of cardiac HYP, left ventricular dysfunction, fibrosis, inflammation and extravasation. Fatty acid metabolism, mitochondrial transcription/translation factors, antioxidant enzymes, and other mitochondrial processes, were downregulated in HF, but not HYP. Proteins upregulated in HF implicate extracellular matrix remodeling, cytoskeletal remodeling, and acute phase inflammation markers. Among metabolites, downregulation of acyl-carnitines was observed in HYP, while fatty acids accumulated in HF. Correlation of transcript and protein changes in HF is weak (R2=0.23), suggesting transcript/proteome divergence may reveal post-transcriptional gene regulation in HF. Proteome/Metabolome integration suggests metabolic bottlenecks in fatty acyl-CoA processing by carnitine palmitoyl transferase (CPT1B) as well as TCA cycle inhibition. We present a model by which acute signaling in HF, including Ca2+ dysregulation and low cAMP levels, is coupled to mitochondrial metabolic and antioxidant defects, through a CREB/PGC1 transcriptional axis. Animal Model The guinea pig model of heart failure and sudden cardiac death has been described previously. Briefly, the HF and SCD guinea pig model was produced by combining ascending aortic constriction (AC) and daily isoproterenol challenge (ACi model). Specifically, Hartley guinea pigs (~250 g; Hilltop Lab Animals) were anesthetized with 4% isoflurane in a closed box for 4min, and then intubated and ventilated with oxygen and 2% isoflurane. Ascending aortic constriction (AC) was produced by tying a suture around the ascending aorta using an 18‐gauge needle as a spacer, which was then removed. For sham operations the procedure was identical though the suture was not tied. After the procedure, bupronex (0.05 mg/kg) was administered via intramuscular injection for analgesia and animals were observed until full recovery. Isoproterenol was administered daily by intra peritoneal injection at 1 mg/kg for the first week after surgery and at 2 mg/kg for a subsequent 3 weeks. As characterized previously (1), cardiac function of ACi animal is well compensated in the first 2 weeks (HYP) but declined rapidly thereafter (HF). Hypertrophic heart was collected between 1-2 weeks post-surgery (ACi-2w), whereas failing heart was collected at 4 weeks after surgery (ACi-4w). Following retrograde perfusion with 20ml Tyrode’s solution, excised hearts were Snap-frozen in liquid N2 and stored at -80°C Experimental Design The experiment consisted of 3 treatment groups: 1) HYP (ACi-2wk), 2) HF (ACi-4wk) 3) sham-operated animals with daily administration for 4 weeks (Shami-4w). 1 heart from each group was included in an iTRAQ 4-plex experiment wherein peptides from each heart are subjected to reaction with an isobaric label. The experiment was repeated twice, yielding a total of 3 independent experiments quantifying the peptides from 9 hearts. ITRAQ reagents were shuffled among treatment groups for each experiment to minimize labeling bias.
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