Production and characterization of a monoclonal antibody specific to Nef-associated factor 1 (Naf1)/A20-binding inhibitor of NF-κB activation (ABIN-1)

Cellular protein Naf1 (Nef-associated factor 1) or ABIN-1 (A20-binding inhibitor of NF-κB activation) is an important cellular protein, expressed in various human tissues and T-cell lines. Naf1 protein has two isoforms (Naf1α and Naf1β) with different C-termini, produced by alternative splicing. Naf1α and Naf1β have approximately 2800 and 2600 nucleotides, with an open reading frame of 1941 and 1781 nucleotides, encoding the 72-kDa Naf1α and 68-kDa Naf1β proteins, respectively. In the present study, we generated a monoclonal antibody (MAb) against human Naf1, which recognizes full-length, endogenous Naf1 of both isotypes. For this purpose, recombinant 6×His and myc-tagged N-terminal Naf138–135, Naf1(N) protein was produced by using the baculovirus expression system. Recombinant Naf1(N) protein was used to immunize Balb/c mice, and a hybridoma cell line producing stable and highly specific MAb with strong affinity to Naf1 was established. We further characterized this antibody by immunofluorescent assay an...