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Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation
Published on: June 27, 2017
Proteomics analysis of immunoprecipitated proteins associated with the oncogenic kinase cot
1School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore.
Abstract:
Cancer Osaka thyroid, also known as Tpl-2 (Cot) is a member of the MAP3K kinase family and plays a key role in the regulation of the immune response to pro-inflammatory stimuli such as lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha). A series of Cot constructs with an N-terminal 6xHis tag were transiently expressed in HEK293 cells: Cot(130-399) (kinase domain), Cot(1-388) (N-terminal and kinase domains), Cot(1-413), Cot(1-438) (containing a putative PEST sequence), Cot(1-457) (containing both PEST and degron sequences) and Cot(1-467) (full-length protein). These Cot proteins were pulled down using an anti-6xHis antibody and separated by 2D electrophoresis. The gels were silver-stained and 21 proteins were detected that did not appear, or had substantially reduced intensity, in the control sample. Three of these were identified by MS and MS/MS analysis as Hsp90, Hsp70 and Grp78. Hsp90 appeared to bind to the kinase domain of Cot and this interaction was further investigated using co-immuno-precipitation with both overexpressed Cot in HEK293 cells and endogenous Cot in Hela cells.
Insights
Cancer Osaka thyroid (Cot), a MAP3K kinase, regulates immune responses. This study identified heat shock proteins Hsp90, Hsp70, and Grp78 interacting with Cot, particularly Hsp90 binding to its kinase domain.
Area of Science:
- Molecular Biology
- Immunology
- Cell Biology
Background:
- Cancer Osaka thyroid (Cot), also known as Tpl-2, is a MAP3K kinase family member.
- Cot plays a crucial role in immune response regulation against pro-inflammatory stimuli like LPS and TNF-alpha.
Purpose of the Study:
- To identify proteins interacting with Cot.
- To investigate the interaction between Cot and heat shock proteins, specifically Hsp90.
Main Methods:
- Transient expression of various Cot constructs (N-terminal 6xHis tagged) in HEK293 cells.
- Protein pull-down using anti-6xHis antibody followed by 2D electrophoresis and silver staining.
- Mass spectrometry (MS and MS/MS) for protein identification and co-immunoprecipitation for interaction validation.
Main Results:
- Twenty-one proteins were detected interacting with Cot constructs.
- Hsp90, Hsp70, and Grp78 were identified as Cot-interacting proteins.
- Hsp90 demonstrated binding to the Cot kinase domain, confirmed by co-immunoprecipitation in HEK293 and Hela cells.
Conclusions:
- Heat shock proteins, including Hsp90, Hsp70, and Grp78, interact with Cancer Osaka thyroid (Cot).
- The kinase domain of Cot is involved in its interaction with Hsp90.
- These findings provide insights into Cot regulation and function within the cellular environment.
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