Procedures

  Antibody production

Antigens were produced by either expressing GST- or GST/6His-fusion polypeptide in E. coli or myc/6His- or FLAG-fusion protein in mammalian cells, or by synthesizing peptide conjugated with keyhole limpet hemocyanin. In most cases, two non-overlapping antigenic fragments were produced for each protein. For producing polyclonal antibodies, two rabbits were immunized repeatedly, and resulting antibodies were affinity-purified from the antisera. Monoclonal antibodies were raised in rats according to Sado et. al. (1995).