ArticlePloS one2013
Effective identification of Akt interacting proteins by two-step chemical crosslinking, co-immunoprecipitation and mass spectrometry.
Article in PloS one, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed, 48 citations in OpenAlex.
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- Improved Immunoprecipitation to Mass Spectrometry Method for the Enrichment of Low-Abundant Protein Targets.Methods in molecular biology (Clifton, N.J.) · 2021Article
- Direct stimulation of ERBB2 highlights a novel cytostatic signaling pathway driven by the receptor ThrScientific reports · 2020Article
- Cinnamaldehyde protects against rat intestinal ischemia/reperfusion injuries by synergistic inhibition of NF-κB and p53.Acta pharmacologica Sinica · 2020Article
- Metabolic syndrome diminishes insulin-induced Akt activation and causes a redistribution of Akt-interacting proteins in cardiomyocytes.PloS one · 2020Article
- Proteomics for cancer drug design.Expert review of proteomics · 2019Article
- EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica.PLoS pathogens · 2019Article
- Protein-protein interactions of the nicotinamide/nicotinate mononucleotide adenylyltransferase of Leishmania braziliensis.Memorias do Instituto Oswaldo Cruz · 2019Article
- AKT/protein kinase B associates with β-actin in the nucleus of melanoma cells.Bioscience reports · 2019Article
- Downregulation of miR-199a-3p mediated by the CtBP2-HDAC1-FOXP3 transcriptional complex contributes to acute lung injury by targetingInternational journal of biological sciences · 2019Article
- Distinct CD40L receptors mediate inflammasome activation and secretion of IL-1β and MCP-1 in cultured human retinal pigment epithelial cells.Experimental eye research · 2018Article
- Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation.The American journal of pathology · 2018Article
- Mapping of Plasma Membrane Proteins Interacting WithFrontiers in plant science · 2018Article
- A Screen for Protein-Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A.ACS infectious diseases · 2017Article
- A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis.Journal of visualized experiments : JoVE · 2017Article
- An overview of technical considerations for Western blotting applications to physiological research.Scandinavian journal of medicine & science in sports · 2017Review
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners.
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