A monoclonal antibody recognizing nestin and the phosphorylated form of neurofilament-M

In the course of an immunohistochemical screening of antibodies raised against a postnatal day 7 rat brain membrane fraction, we obtained a monoclonal antibody that recognizes both neurofilament-M, a type VI intermediate filament protein, and nestin, a type IV intermediate filament protein. In newborn rat cerebellum, the 5A7 antibody stained Bergmann glial fibers and subsets of axons simultaneously, but in adult cerebellum it only stained neuronal elements. Upon immunoblot analysis of neonatal rat cerebellum, 5A7 gave two prominent bands corresponding to 140 and > 200 kDa. The former was identified as neurofilament-M and the latter as nestin. Dephosphorylation of neurofilament-M resulted in a dramatic reduction in the immunoreactivity, indicating that 5A7 recognized the phosphorylated form of neurofilament-M. Immunoblot analysis of V8 protease digests of neurofilament-M revealed that the epitope on neurofilament-M is located on the carboxy-half of the tail domain of neurofilament-M. These results indicate that 5A7 is the first monoclonal antibody which recognizes a common or similar epitope on different classes of intermediate filament proteins other than the conserved rod domain. The presence of a common or similar epitope on both nestin and neurofilament-M recognized by 5A7 may suggest the possibility that nestin and neurofilament-M have a common functional motif.