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Preparation and characterization of monoclonal antibodies against GAM protein: a novel gp130-associated molecule
1Department of Immunology, The 4th Military Medical University, Xi'an, P.R. China.
Insights
Monoclonal antibodies (MAbs) were developed to study gp130-associated-molecule (GAM), a protein involved in gp130 and Notch signaling pathways. These MAbs enable detection and characterization of GAM, including its nuclear polymerization.
Area of Science:
- Molecular Biology
- Immunology
- Cell Signaling
Background:
- gp130-associated-molecule (GAM) is a novel protein with homology to Groucho/TLE co-repressors.
- GAM interacts with gp130 and influences JAKs association and tyrosine phosphorylation.
- Understanding GAM's role in signaling pathways like Notch is crucial.
Purpose of the Study:
- To develop high-affinity monoclonal antibodies (MAbs) against GAM.
- To utilize these MAbs for detecting and characterizing GAM in cellular contexts.
- To investigate GAM's function in gp130 and Notch signaling.
Main Methods:
- Expression and purification of GAM-Thioredoxin (Thio) fusion protein.
- Generation of anti-GAM MAbs using hybridoma technology.
- Detection of GAM using Western blotting and flow cytometry; analysis of GAM polymerization.
Main Results:
- Three high-affinity MAbs recognizing distinct GAM epitopes were successfully generated.
- MAbs confirmed GAM expression and detected increased GAM in transfected cells.
- Western blotting revealed GAM forms 110 kDa polymers in the nucleus.
Conclusions:
- The developed MAbs are effective tools for studying GAM.
- These MAbs facilitate investigation into GAM's role in gp130 and Notch signaling.
- GAM polymerization in the nucleus suggests a novel regulatory mechanism.
Abstract:
gp130-associated-molecule (GAM) is a recently cloned 24-kDa protein, which binds to gp130 at its cytoplasmic membrane-proximal region and has high homology with the N-terminal of Groucho/TLE molecules, a transcription co-repressor family playing an essential role in Notch signaling. Expression of GAM in COS7 cells inhibited the association of JAKs with gp130, and decreased the tyrosine phosphorylation level of these molecules as well. To further investigate the function of GAM, monoclonal antibodies (MAbs) to GAM were prepared. First, GAM-Thioredoxin(Thio) fusion protein was expressed in E. coli and purified with anti-Thio PAb coupled Sepharose-4B. Using purified GAM-Thio as immunogen, three MAbs against GAM with high affinity were raised by conventional B-lymphocyte hybridoma technique. They could recognize different epitopes of nature and denatured GAM-Thio without any cross-reaction with Thio or components of E. coli or with TLE1-GST fusion protein. In Western blotting and flow cytometric assay, these MAbs can detect cellular GAM protein and verify the increase of GAM expressing in GAM cDNA permanently transfected M1 cells. Furthermore, Western blotting with these MAbs indicated that GAM formed 110 kDa polymers in the nucleus. These MAbs represent powerful in investigating the role of GAM in gp130 signaling and Notch signaling.
