Preparation and characterization of monoclonal antibodies against GAM protein: A novel gp130-associated molecule

gp130-associated-molecule (GAM) is a recently cloned 24-kDa protein, which binds to gp130 at its cytoplasmic membrane-proximal region and has high hom ology with the N-terminal of Groucho/TLE molecules, a transcription co-repr essor family playing an essential role in Notch signaling. Expression of GA M 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 p repared. First, GAM-Thioredoxin(Thio) fusion protein was expressed in E. co li and purified with anti-Thio PAb coupled Sepharose-4B. Using purified GAM -Thio as immunogen, three MAbs against GAM with high affinity were raised b y conventional B-lymphocyte hybridoma technique. They could recognize diffe rent epitopes of nature and denatured GAM-Thio without any cross-reaction w ith Thio or components of E. coli or with TLE1-GST fusion protein, In Weste rn blotting and flow cytometric assay, these MAbs can detect cellular GAM p rotein and verify the increase of GAM expressing in GAM cDNA permanently tr ansfected M1 cells, Furthermore, Western blotting with these MAbs indicated that GAM formed 110 kDa polymers in the nucleus. These MAbs represent powe rful in investigating the role of GAM in gp130 signaling and Notch signalin g.