The Immunogen: a-internexin is a Class IV intermediate filament originally discovered as it copurifies with other neurofilament subunits (1). On SDS-PAGE gels it runs with an apparent molecular weight of 64 to 66 kDa, with some species variability, although the real molecular weight is about 55kDa; as with the other neurofilament subunits the presence of highly negatively charged sequences results in reduction of SDS-PAGE mobility. a-internexin is related to but distinct from the better known neurofilament triplet proteins, NF-L, NF-M and NF-H, having similar protein sequence motifs and a similar intron organization. It is expressed only in neurons and in large amounts early in neuronal development, but is down-regulated in many neurons as development procedes. Many classes of mature neurons contain a-internexin in addition to NF-L, NF-M and NF-H. In some mature neurons a-internexin is the only neurofilament subunit expressed. Antibodies to a-internexin are therefore unique probes to study and classify neuronal types and follow their processes in sections and in tissue culture. In addition the very early developmental expression of a-internexin means its presence is an early and convenient diagnostic feature of neuronal progenitors cells and other cell committed to the neuronal lineage. In addition recent studies show a marked up-regulation of a-internexin during neuronal regeneration (2). The use of antibodies to this protein in the study of brain tumors has not been examined to date, but is likely to be of interest. Recently Trojanowski, Lee and coworkers used this antibody to show that a-internexin is an abundant component of the inclusions of neurofilament inclusion body disease (NFID, see two Cairns et al. papers listed below), a serious human neurodegenerative disorder. The antibody was also used to confirm the presence of circulating auto-antibodies to a-internexin in the sera of some patients with endocrine autoimmunity, as well as in some normal individuals (Rajasalu et al., 2004, listed below). The HGNC name for this protein is INA.

Left: blot of homogenate of rat facial nucleus incubated with MCA-2E3, which reveals a single clean band running at ~66kDa. Right: Rat facial nucleus before (left) and after (right) facial nerve lesion. Note the profound upregulation of a-internexin in the facial nucleus motor neurons.

Antibody characteristics: MCA-2E3 was raised against recombinant rat a-internexin expressed in and purified from E. coli (3). MCA-2E3 reacts with human a-internexin and with this protein in all mammalian species tested to date. On Western blots the antibody is unusually strong and specific in bindingas shown above. The antibody is clean and specific on immunoblots. Can be used on formalin-fixed cells in tissue culture, cryostat sections, and Western blotting. The epitope recognized by the 2E3 clone is in the C-terminal non-helical extension of the protein and is unusually resistant to aldehyde fixation, so that this antibody is ideal for studies of paraffin embedded formalin fixed histological sections.

Suggestions for use: For immunofluorescence try dilutions of 1:500, for immunohistochemistry with ABC or other enzymatic amplification procedures try 1:5,000. For western blots try 1:10,000 dilutions.

Limitations: This product is for research use only and is not approved for use in humans or in clinical diagnosis.

References:

1. Pachter, J and Liem, RKH. Alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues. J Cell Biol 101:1316-22 (1985)

2. McGraw et al. Axonally transported peripheral signals regulate alpha-internexin expression in regenerating motoneurons. J Neurosci 22:4955-63 (2002)

3. Evans J. et al. Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem. J. Neurophysiol. 87:1076-85 (2002).

Recent Papers using this antibody:

1. Ko TL, Chien CL, Lu KS. The expression of alpha-internexin and peripherin in the developing mouse pineal gland. J Biomed Sci. 2005 Aug 19 :1-13 [Epub ahead of print] .
2. Wang P, Wang SM, Hsieh CJ, Chien CL. Neural expression of alpha-internexin promoter in vitro and in vivo. J Cell Biochem. 2005 Sep 19 [Epub ahead of print].
3. Rauch U, Klotz M, Maas-Omlor S, Wink E, Hansgen A, Hagl C, Holland-Cunz S, Schafer KH. Expression of Intermediate Filament Proteins and Neuronal Markers in the Human Fetal Gut. J Histochem Cytochem. 2005 Aug 8; [Epub ahead of print] .
4. Uchikado H, Shaw G, Wang DS, Dickson DW. Screening for neurofilament inclusion disease using alpha-internexin immunohistochemistry. Neurology. 64:1658-9 (2005).
5. Chien CL, Liu TC, Ho CL, Lu KS. Overexpression of neuronal intermediate filament protein alpha-internexin in PC12 cells. J Neurosci Res. 80:693-706 (2005).
6. Jing-Ping Z, Tian QB, Sakagami H, Kondo H, Endo S, Suzuki T. p55 protein is a member of PSD scaffold proteins in the rat brain and interacts with various PSD proteins. Brain Res Mol Brain Res. 135:204-16 (2005).
7. Josephs KA, Uchikado H, McComb RD, Bashir R, Wszolek Z, Swanson J, Matsumoto J, Shaw G, Dickson DW Extending the clinicopathological spectrum of neurofilament inclusion disease. Acta Neuropathol (Berl). 109:427-32 (2005).
8. Cairns NJ et al. Clinical and neuropathologic variation in neuronal intermediate filament inclusion disease. Neurology. 63:1376-84 (2004).
9. Rajasalu T, Teesalu K, Janmey PA, Uibo R. Demonstration of natural autoantibodies against the neurofilament protein alpha-internexin in sera of patients with endocrine autoimmunity and healthy individuals. Immunol Lett. 94:153-60 (2004).
10. Cairns NJ et al. alpha-Internexin aggregates are abundant in neuronal intermediate filament inclusion disease (NIFID) but rare in other neurodegenerative diseases. Acta Neuropathol (Berl). May 28 [Epub ahead of print] (2004).
11. Cairns NJ et al. alpha-internexin is present in the pathological inclusions of neuronal intermediate filament inclusion disease. Am J Pathol. 164:2153-61 (2004).
12. Yuan A, Rao MV, Kumar A, Julien JP, Nixon RA. Neurofilament transport in vivo minimally requires hetero-oligomer formation. J Neurosci. 23:10463-10475 (2003).
13. Ortino B, Inverardi F, Morante-Oria J, Fairen A, Frassoni C. Substrates and routes of migration of early generated neurons in the developing rat thalamus. Eur J Neurosci. 18:323-32 (2003).
14. Stachowiak EK, Fang X, Myers J, Dunham S, Stachowiak MK. cAMP-induced differentiation of human neuronal progenitor cells is mediated by nuclear fibroblast growth factor receptor-1 (FGFR1). J Neurochem. 84:1296-312 (2003).
15. Donner LR, Teshima I. Peripheral medulloepithelioma: an immunohistochemical, ultrastructural, and cytogenetic study of a rare, chemotherapy-sensitive, pediatric tumor. Am J Surg Pathol. 27:1008-1012 (2003).
16. Ortino B, Inverardi F, Morante-Oria J, Fairen A, Frassoni C. Substrates and routes of migration of early generated neurons in the developing rat thalamus. Eur J Neurosci. 18:323-32 (2003).
17. Larivière, R. C., Nguyen, M. D., Ribeiro-da-Silva, A. and Julien J.-P. Reduced number of unmyelinated sensory axons in peripherin null mice. J. Neurochem. 81:525-532 (2002)
18. Talbot NC, Powell AM, Garrett WM. Spontaneous differentiation of porcine and bovine embryonic stem cells (epiblast) into astrocytes or neurons. In Vitro Cell Dev Biol Anim. 38:191-197 (2002).
19. Shaw G, Morse S, Ararat M, Graham FL. Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J. 16:869-71 (2002).

Examples: For image of an immunoblot generated with this antibody press here. For some immunocytochemical images generated with this antibody press here

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