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Journal of Neuroscience, Vol 7, 3474-3488, Copyright © 1987 by Society for Neuroscience
Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats
VM Lee, MJ Carden, WW Schlaepfer and JQ Trojanowski
Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical School, Philadelphia 19104.
A new panel of greater than 300 monoclonal antibodies (mAbs) was prepared
to the high, middle, and low Mr rat neurofilament (NF) subunits (NF-H, NF-M
and NF-L, respectively). NF proteins were purified both from native, i.e.,
phosphorylated rat NFs and from enzymatically dephosphorylated rat NFs. The
resulting mAbs were used to biochemically and immunochemically distinguish
and characterize distinct and differentially phosphorylated isoforms of NF
subunits. By immunoblot, all mAbs specific for NF-L and some mAbs specific
for NF-M detected their specific NF subunit regardless of whether or not
the NFs had been treated with alkaline phosphatase, and such antibodies
were termed "phosphate-independent" or P[ind] mAbs. The other mAbs were
specific for NF-M, NF-H, or for both NF-M and NF-H, and they recognized
epitopes in the COOH termini of these subunits. Significantly, the latter
mAbs could discriminate different isoforms of NF-M and NF-H, depending on
the phosphorylation state of each variant. Such mAbs were assigned to one
of 4 distinct categories on the basis of their performance in immunoblots
of progressively dephosphorylated rat NF samples and by
immunohistochemistry of various adult rat nervous tissues: (1) P[-] mAbs
preferentially stained neuronal perikarya and dendrites, and they
recognized only extensively dephosphorylated (and nonphosphorylated) NF- H;
(2) P[+] mAbs stained axons more strongly than perikarya, and primarily
blotted phosphorylated, but not nonphosphorylated, forms of NF-H and NF-M;
(3) P[++] mAbs stained axons almost to the exclusion of perikarya, and in
blots recognized only the extensively phosphorylated forms of NF-H and NF-M
(i.e., subunits subjected to limited enzymatic dephosphorylation); (4) P[ ]
mAbs also predominantly stained axons, but the briefest alkaline
phosphatase treatment abolished the NF-M and NF-H immunobands produced by
these mAbs. Two-dimensional gel analysis and immunoblotting of total
proteins from adult rat dorsal root ganglion verified mAb specificity in
situ, and showed that differentially phosphorylated isoforms of NF-M and
NF-H occur in vivo. This provided additional evidence that mAbs can detect
all 4 phosphorylation- dependent endogenous isoelectric variants of NF-H
and NF-M.(ABSTRACT TRUNCATED AT 400 WORDS)
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