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European journal of cell biology

Reactivity of a panel of neurofilament antibodies on phosphorylated and dephosphorylated neurofilaments.


PMID 3539605

Abstract

The work of the Sternbergers and their colleagues has shown that monoclonal antibodies reactive with neurofilament subunit proteins may be sensitive to the state of phosphorylation of these proteins. We therefore examined the ability of our previously described panel of monoclonal and polyclonal neurofilament antibodies to bind to normal and to enzymatically dephosphorylated neurofilament subunits. All the monospecific antibodies, both mono- and polyclonal, which we had previously documented as reactive with neurofilament H protein proved to bind only to the phosphorylated form of this protein, and H antibody staining of neurofilamentous profiles in frozen sections could be abolished by appropriate pretreatment of sections with alkaline phosphatase. In contrast, all monospecific antibodies, both mono- and polyclonal, reactive with native M and L proved to bind with apparently undiminished affinity following enzymatic dephosphorylation of the appropriate antigen, either in frozen sections or on Western blots. The class of monoclonal antibodies which react with both H and M were variable in their response to dephosphorylated neurofilaments; some completely lost their reactivity whilst others were partially or wholly unaffected. We stained frozen sections of nervous tissues from various mammalian species with the panel of antibodies, and observed filamentous staining of the perikarya and dendrites of a variety of different types of neuron with all antibodies, both mono- and polyclonal, directed against L and M. Antibodies with strong reactivity for phosphorylated H always failed to stain neurofilamentous dendritic and perikaryal profiles. We further describe the isolation and characterization of a new monoclonal antibody, which recognizes both phosphorylated and enzymatically dephosphorylated forms of H.(ABSTRACT TRUNCATED AT 250 WORDS)