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Journal of Neuroscience, Vol 5, 2779-2785, Copyright © 1985 by Society for Neuroscience

ARTICLE

Immunoblot analysis of horseradish peroxidase conjugates of wheat germ agglutinin before and after retrograde transport in the rat peripheral nervous system

ML Schmidt and JQ Trojanowski

Immunoblot studies were performed on tissue extracts of rats injected with horseradish peroxidase (HRP) conjugates of wheat germ agglutinin (WGA) using anti-HRP and anti-WGA antisera. Immunoblots of uninjected, purified conjugates (WGA-HRP) demonstrated a 62-kilodalton (kd) conjugate (monomeric WGA conjugated to HRP). Other prominent immunobands included HRP (40 kd), monomeric WGA (22 kd), and 15- to 30- kd HRP breakdown products. Following injections of WGA-HRP into the submandibular gland of rats which survived 16 hr to 8 days, immunoblots were performed on homogenates of injected submandibular glands and of superior cervical ganglia containing neurons retrogradely labeled with WGA-HRP. The anti-HRP antiserum detected unconjugated, 40-kd HRP and a 62-kd immunoband corresponding to WGA-HRP in the superior cervical ganglia and submandibular glands. No immunobands were detected with the antiserum to WGA in superior cervical ganglia homogenates. Blots of submandibular gland homogenates harvested 24 hr after a WGA-HRP injection, but not at later time points, contained an immunoband reactive with the anti-WGA antiserum; it corresponded to monomeric WGA. These studies analyze for the first time the molecular composition of WGA-HRP conjugates before and after retrograde transport; they provide a novel approach for probing the intraneuronal transport and degradation of proteins. We conclude that morphologically defined endocytic pathways using protein markers reflect the endocytosis and transport of immunochemically altered and unaltered forms of the markers.

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