Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Determination of Rab5 activity in the cell by effector pull-down assay.

ABSTRACT: Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.

SUBMITTER: Qi Y

PROVIDER: S-EPMC5901726 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Determination of Rab5 activity in the cell by effector pull-down assay.

Qi Yaoyao Y Liang Zhimin Z Wang Zonghua Z Lu Guodong G Li Guangpu G

Methods in molecular biology (Clifton, N.J.) 20150101

Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we ...[more]

PMID: 25800849

Similar Datasets

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

Project description:ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved Adenylate-Uridylate-rich Elements (AREs) located in 3' untranslated regions (UTRs) to mediate RNA decay. We hypothesized that ZFP36L1 is a negative regulator of a post-transcriptional hub involved in the RNA half-life regulation of cancer-related transcripts. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo; whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wildtype and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells ± tet-on ZFP36L1, was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1, including HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3' UTRs of these targets for RNA degradation, thus suppressing their expression. Collectively, our findings reveal an indispensable role of ZFP36L1 as a post-transcriptional safeguard against aberrant hypoxic signaling and abnormal cell cycle progression.

2020-10-02 | GSE136179 | GEO

Facile single-molecule pull-down assay for analysis of endogenous proteins.

Project description:The single-molecule pull-down (SiMPull) assay analyzes molecular complexes in physiological conditions from cell or tissue lysates. Currently the approach requires a lengthy sample preparation process, which has largely prevented the widespread adoption of this technique in bioanalysis. Here, we present a simplified SiMPull assay based upon dichlorodimethylsilane-Tween-20 passivation and F(ab) fragment labeling. Our passivation is a much shorter process than the standard polyethylene glycol passivation used in most single-molecule studies. The use of F(ab) fragments for indirect fluorescent labeling rather than divalent F(ab')2 or whole IgG antibodies allows for the pre-incubation of the detection antibodies, reducing the sample preparation time for single-molecule immunoprecipitation samples. We examine the applicability of our approach to recombinant proteins and endogenous proteins from mammalian cell lysates.

| S-EPMC9238377 | biostudies-literature

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation.

Project description:Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

| S-EPMC9317552 | biostudies-literature

Analysis on the interaction domain of VirG and apyrase by pull-down assay.

Project description:VirG is outer membrane protein of Shigella and affects the spread of Shigella. Recently it has been reported that apyrase influences the location of VirG, although the underlying mechanism remains poorly understood. The site of interaction between apyrase and VirG is the focus of our research. First we constructed recombinant plasmid pHIS-phoN2 and pS-(v1-1102, v53-758, v759-1102, v53-319, v320-507, v507-758) by denaturation-renaturation, the phoN2:kan mutant of Shigella flexneri 5a M90T by a modified version of the lambda red recombination protocol originally described by Datsenko and Wanner and the complemented strain M90TΔphoN2/pET24a(PhisphoN2). Second, the recombinant plasmid pHIS-phoN2 and the pS-(v1-1102, v53-758, v759-1102, v53-319, v320-507, v507-758) were transformed into E. coli BL21 (DE3) and induced to express the fusion proteins. Third, the fusion proteins were purified and the interaction of VirG and apyrase was identified by pull-down. Fourth, VirG was divided and the interaction site of apyrase and VirG was determined. Finally, how apyrase affects the function of VirG was analyzed by immunofluorescence. Accordingly, the results provided the data supporting the fact that apyrase combines with the α-domain of VirG to influence the function of VirG.

| S-EPMC6271496 | biostudies-literature

RNA pull-down assay

Project description:To detect RNA binidng proteins bound with SynGAP 3'UTR, the biotinylated RNA probe was generated, and the bands were detected by silver staining.

2018-08-21 | PXD007521 | JPOST Repository

Pull-Down Assay-Guided Insights into the Effects of Latroeggtoxin-VI on Nerve Cells.

Project description:Latroeggtoxin-VI (LETX-VI) is a peptide neurotoxin newly found from the eggs of spider L. tredecimguttatus. To explore the mechanism of action of the LETX-VI on nerve cells, the effects of LETX-VI on PC12 cells, a commonly used neuron model, were analyzed using a pull-down assay-guided strategy. LETX-VI was shown to interact with 164 PC12 cell proteins that have diverse molecular functions such as binding, catalysis, regulation, structural activity, etc., thereby extensively affecting the biological processes in the PC12 cells, particularly protein metabolism, response to stimulus, substance transport, and nucleic acid metabolism, with 56.71%, 42.07%, 29.88% and 28.66% of the identified proteins being involved in these biological processes, respectively. By interacting with the relevant proteins, LETX-VI enhanced the synthesis of dopamine; positively regulated cell division and proliferation; and negatively regulated cell cycle arrest, cell death, and apoptotic processes, and therefore has limited cytotoxicity against the PC12 cells, which were further experimentally confirmed. In general, the effects of LETX-VI on PC12 cells are more regulatory than cytotoxic. These findings have deepened our understanding of the action mechanism of LETX-VI on nerve cells and provided valuable clues for further related researches including those on Parkinson's disease.

| S-EPMC7918074 | biostudies-literature

Protocol for detecting lncRNA-protein interactions in vitro by tRSA RNA pull-down assay.

Project description:Long non-coding RNAs (lncRNAs) work together with diverse RNA-binding proteins (RBPs) to fulfill key regulations in important cellular functions. Here, we present a protocol to detect lncRNA-RBP interactions in vitro using a tRNA scaffold containing a streptavidin aptamer pull-down assay. We describe steps for preparing both protein and lncRNA transcripts, lncRNA-protein interaction detection with an in vitro binding assay, and western blot analysis. This protocol is applicable to screen for RNA-interacting proteins using cell lysates followed by mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Yang et al. (2023).1.

| S-EPMC10789616 | biostudies-literature

RNA-biotin based pull down assay with Hela Nuclear Extract followed by RNA-seq

Project description:We aimed to discover trans-acting RNA molecules involved in mRNA 3’ processing. We reasoned that, if there exist such functional RNAs, they must directly associate with the key machinery responsible for mRNA 3’ processing. Therefore, it would be of great value to comprehensively identify RNAs interacting with pre-mRNA 3’ processing complex. To this goal, we took advantage of previously well-characterized system combined with high-throughput sequencing to investigate the target RNAs at the transcriptomic level. Briefly, we used polyA site (PAS) RNA substrates, SV40 late (SVL), and corresponding control RNAs with point mutation (U to C) at the highly conserved cis-element AAUAAA. RNA substrates were first biotinylated at the 3’ end, and then bound to the streptavidin magnetic beads. After incubation with Hela Nuclear Extract (NE) under polyadenylation condition, the two wild type RNA substrates could specifically recruit mRNA 3’ processing factors in NE for complex assembly. The purification of the protein complex and its interacting RNAs were performed using biotin-streptavidin pull-down. We extracted RNAs from the pull-down sample, prepared the strand-specific RNA-Seq libraries and submitted them for deep-sequencing.

2017-07-01 | GSE98218 | GEO

Proof of concept: Pull down assay using bovine serum albumin and an immunomodulator small molecule.

Project description:In the past decade, there has been increasing interest in use of small molecules for immunomodulation. The affinity-based pull-down purification is an essential tool for target identification of small molecules and drug discovery. This study presents our recent efforts to investigate the cellular target(s) of Compound A, a small molecule with demonstrated immunomodulatory properties in human peripheral blood mononuclear cells (PBMCs). While we have previously observed the immunomodulatory activity of Compound A in PBMCs, the specific molecular targets underlying its effects remains elusive. To address this challenge, we synthesized a trifluoromethyl phenyl diazirine (TPD)-bearing trifunctional Probe 1 based on the chemical structure of Compound A, which could be used in a pull-down assay to efficiently bind to putative cellular targets via photoaffinity labelling. In this report, we utilized bovine serum albumin (BSA) as a model protein to establish a proof-of-concept in order to assess the suitability of Probe 1 for binding to an endogenous target. By the successful synthesis of Probe 1 and demonstrating the efficient binding of Probe 1 to BSA, we propose that this method can be used as a tool for further identification of potential protein targets of small molecules in living cells. Our findings provide a valuable starting point for further investigations into the molecular mechanisms underlying the immunomodulatory effects of Compound A.

| S-EPMC10651465 | biostudies-literature

Evidence that the rab5 effector APPL1 mediates APP-βCTF-induced dysfunction of endosomes in Down syndrome and Alzheimer's disease.

Project description:β-Amyloid precursor protein (APP) and its cleaved products are strongly implicated in Alzheimer's disease (AD). Endosomes are highly active APP processing sites, and endosome anomalies associated with upregulated expression of early endosomal regulator, rab5, are the earliest known disease-specific neuronal response in AD. Here, we show that the rab5 effector APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif) mediates rab5 overactivation in Down syndrome (DS) and AD, which is caused by elevated levels of the β-cleaved carboxy-terminal fragment of APP (βCTF). βCTF recruits APPL1 to rab5 endosomes, where it stabilizes active GTP-rab5, leading to pathologically accelerated endocytosis, endosome swelling and selectively impaired axonal transport of rab5 endosomes. In DS fibroblasts, APPL1 knockdown corrects these endosomal anomalies. βCTF levels are also elevated in AD brain, which is accompanied by abnormally high recruitment of APPL1 to rab5 endosomes as seen in DS fibroblasts. These studies indicate that persistent rab5 overactivation through βCTF-APPL1 interactions constitutes a novel APP-dependent pathogenic pathway in AD.

| S-EPMC4721948 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data