Project description:In this study, pull down assays combined with mass spectrometry analysis were performed in HL-1 cardiomyocytes transfected with control plasmid (OE-Vector) or LOC107984012-overexpression plasmid to identify the repertoire of LOC107984012 interacting proteins.
Project description:Mass spectrometry analysis of proteins interacting with STAT3¬Flag was performed. STAT3-Flag was transiently expressed in HeLa cells and the interactome of STAT3-Flag was studied in lysosomal extracts. proteins co-¬immunoprecipitating with STAT3--Flag identified eight subunits of the vacuolar H+-¬ ATPase (V-¬ATPase) as potential STAT3 binding partners. Validation experiments were performed by antibody pull-down of STAT3.
Project description:dentification of LAGE3-Interacting Proteins Under DNA Replication Stress via Flag-LAGE3 Pull-Down Coupled with Mass Spectrometry Analysis
Project description:In this study, pull down assays combined with mass spectrometry analysis were performed in AC16 cardiomyocytes transfected with control plasmid (OE-Vector) or LOC107984012-overexpression plasmid to identify the repertoire of LOC107984012 interacting proteins. Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) provides reagents to efficiently enrich RNA Binding Proteins. Sense or antisense of LOC107984012 were in vitro transcription using the T7 RNA transcription system (Large Scale RNA ProductionSystem-T7, Promega) and biotin-labeled with the Pierce™ RNA 3’ End Desthiobiotinylation Kit (Thermo Scientific). RNA was then purified with QIAquick PCR Purification Kit (Qiagen). 30 μg of whole-cell protein lysates were incubated with purified biotinylated transcripts for 1 h at 4°C with rotation, then they were eluted for mass spectrometry identification.
Project description:To dissect the molecular mechanisms of HILPS in promoting cell survival under hypoxia, we conducted RNA pull-down assay in combination with mass spectrometry analysis to identify HILPS-interacting proteins. Pull-down of antisense HILPS was used as a negative control. To identify the potential phosphorylation site(s), we conducted the in vitro kinase assay in combination with mass spectrometry analysis using PLK1-T210D, recombinant GST-HIF1a and cold ATP. A kinase assay using PLK1-K82R was included as a control.
Project description:To identify the potential interacting protein of LILRB1 in multiple myeloma cell line ARP-1,we used anti-IgG antibody and anti-LILRB1 antibody to do pull down in ARP-1 cell lysate and sent the pull-down products for mass spectrum analysis.By comparing the pull-down results of anti-LILRB1 antibody and anti-IgG antibody, we identify potential interacting proteins of LILRB1.
Project description:Identification of interacting partners of the Partner and Localizer of BRCA2 (PALB2), essential regulator of DNA repair by homologous recombination. Interacting partners of PALB2 were purified by GFP pull down from asynchronous HEK293 Flp-In T-REx cells, exogenously expressing Flag-EGFP tagged PALB2 at a similar level than the endogenous PALB2 level. Before GFP pull down, whole cell protein extracts were pre-cleared on IgG agarose beads to decrease binding of non-specific proteins during GFP pull down. GFP pull down were performed using GFP-Trap agarose beads (Chromotek) and washed at low salt concentration (150mM NaCl), to maintain interactions of low affinity binding protein partners. As a negative control, Flag-EGFP interacting proteins were purified, following the same protocol as described for the purification of Flag-EGFP PALB2 interacting proteins. Experiments were performed in triplicate.
Project description:Increasing studies show that long non-coding RNAs (lncRNAs) play essential roles in various fundamental processes. Long non-coding RNA growth arrest-specific transcript 5 (GAS5) showed differential expressions between young and old mouse brains in our previous RNA-Seq data, suggesting its potential role in senescence and brain aging. Examination using RT-qPCR revealed that GAS5 had a significantly higher expression level in old mouse brain hippocampus region than the young one. Cellular fractionation using hippocampus-derived HT22 cell line confirmed its nucleoplasm and cytoplasm subcellular localization. We then performed overexpression (OE) or knockdown (KD) of GAS5 in HT22 cell line and found that GAS5 inhibits cell cycle progression and promotes cell apoptosis. RNA-Seq analysis of GAS5-knockdown HT22 cell identified differential expressed genes related to cell proliferation (e.g., DNA replication and nucleosome assembly biological processes). RNA pull-down assay using mouse brain hippocampus tissues revealed that potential GAS5 interacting proteins can be enriched into several KEGG pathways and some of them are involved in senescence associated diseases such as Parkinson and Alzheimer's Disease. These results contribute to better understand the underlying functional network of GAS5 and its interacting proteins in senescence at brain tissue and brain-derived cell line levels. Our study may also provide reference for developing diagnostic and clinic biomarkers of GAS5 in senescence and brain aging.
Project description:To identify which E3 ligases ubiquitinate PD-L1, we overexpressed PD-L1-Flag in HEK293 cells and used anti-Flag magnetic beads to pull down PD-L1 from cell lysates. The interacting proteins were then analyzed by mass spectrometry, revealing that three E3 ligases interact with PD-L1.
Project description:There are several methods to bring down the bait and interactors from the cell lysates, each with its own advantages and disadvantages. Recently, the most commonly used method is one step AP, TAP and proximity-labeling. In order to analyze the Strengths and weaknesses of the three methods, fusion proteins were constructed by NICD4 with FLAG, SFB (S tag-2x Flag tag-SBP tag) and TurboID respectively, NICD4 and its interactors were obtained by purification. By analyzing these proteins, we believe that SFB-TAP is the most reliable and effective method to identify interacting proteins.