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Figure/. Immunofluorescence
analysis ofmAb binding to the
olfactory epithelium. The antibodies
used were: (a, c, and
d) mAb 9-OE; (fand h) mAb
13-OE; (i) mAb 5-OE; and
(j) normal mouse serum as a
negative control, b, e, and g
are phase micrographs of a, d,
and f, respectively. The photographic
and printing conditions
were identical for all
antibodies. Arrows point to
basal cell layer. Stars label olfactory
nerve bundles in the
lamina propria, bg, Bowman's
glands. Bar, 50/zm.
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Figure 2. Histochemical analysis
of SBA and antibody reactivity
in the olfactory bulb.
All panels are coronal sections
through the right olfactory
bulb; lateral is to the
right, dorsal is to the top, and
ventral is to the bottom of
each section. Sections were
labeled with SBA-HRP (a and
b), SBA-HRP and mAb 9-OE
(c and d), mAb 13-OE (e),
mAb 5-OE (f and i), normal
mouse serum (g), and mAb
5A5 (h and j). a and c are micrographs
of the entire right
olfactory bulb. (b and d-h) are
higher power micrographs of
ventral olfactory bulb laminae.
The SBA-HRP-Iabcled
sections in a and b have been
counter-stained with cresyl violet.
i and j are enlargements
of mitral cells and their dendrites,
which are marked by
arrows infand h, respectively.
Arrowheads mark blood vessels
that in some cases contain
blood cells not cleared by perfusion
and therefore stained
nonspecifically. Bars: (a and
c) 500 #m; (b and d-f) 100
/~m; q and i) 50/~m.
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Figure 3. Direct immunoblots of membrane extracts of olfactory
epithelium (a), olfactory bulb (b), and brain (c). Individual lanes
(150/~g membrane protein) were labeled with (lanes a-g, respectively)
mAb 13-OE, raAb 5-OE, mAb 9-OE, normal mouse serum,
SBA-HRP, SBA-HRP plus 0.2 M N-acetyl-D-galactosamine, and
anti-N-CAM 161 sera. In olfactory epithelium, both mAb 5-OE
and 9-OE produced a diffuse background stain throughout the
whole lane. No such labeling occurred in controls when normal
mouse serum was used with either goat anti-mouse IgM (lane d)
or IgG (data not shown) as the second antibody. In general, comparisons
of the density of labeled bands between lanes are not indicative
of relative protein levels due to differences in development
time of the color reagents and to the use of antibodies of different
affinities and avidities (see Results). Qualitative comparisons can
only be made between lanes when the same primary antibody is
used since attempts were made to standardize procedures in these
cases (see Materials and Methods).
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Figure 4. Immunoblots of N-CAM immunoprecipitated
with 161 from olfactory bulb and brain
(each lane represents antigen obtained from 150
#g membrane protein). Antibodies used were
(lanes a-e, respectively) 161, normal rabbit sera,
mAb 13-OE, mAb 5-OE, and mAb 9-OE. 161
weakly labeled a 160-kD band in lane a in olfactory
bulb. The same band was also labeled by
mAbs 13-OE (lane c) and 5-OE (lane d). A similar
weakly stained band was observed in direct immunoblots
of olfactory bulb using 161 (Fig. 3 b, lane
g) and mAbs 13-OE (Fig. 3 b, lane a) and 5-OE
(Fig. 3 b, lane b). The nature of this band is unknown.
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Figure 5. Immunoblots of mAb 9-OE immunopreeipitated antigen
from olfactory bulb (each lane represents antigen obtained from
150 ~tg membrane protein). Lanes a-i, respectively, were reacted
with mAb 9-OE, normal mouse sera, mAb 13-OE, mAb 5-OE,
161, anti-HNK-1, mAb 2B8, anti-L1, and SBA-HRP. The labeled
bands at the bottom of the blots represent background immunoreactivity.
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Figure 6. (a) Complete removal of antigens from olfactory bulb by immunoabsorption. Immunoblots of the initial (lane a) and final (lane
b) sequential mAb 13-OE immunoprecipitation (each lane equivalent to antigen obtained from 150 #g membrane protein) reacted with
mAb 13-OE to show the complete removal of this antigen from olfactory bulb preparations. Similarly, the initial (lane c) and final (lane
d) immunoprecipitation of olfactory bulb with 161 were reacted with 161 to show depletion of this antigen. (b) Cross immunoprecipitation
of antigen depleted preparations. Next, the mAb 13-OE-negative olfactory bulb preparation was both immunoprecipitated and immunoblotted
with 161 (lane a). Controls were sequentially immunoabsorbed with normal mouse sera and then subsequently both immunoprecipitated
and immunoblotted with 161 (lane b). The 161-negative olfactory bulb preparation was both immunoprecipitated and immunoblotted with
mAb 13-OE (lane c). In this case, controls were sequentially immunoabsorbed with normal rabbit sera and then both immunoprecipitated
and immunoblotted with mAb 13-OE (lane d). The labeled N-CAM bands in control lanes revealed that antigens survive intact the rigorous
sequential immunoprecipitation protocol. (c) mAb 9-OE immunoprecipitation of N-CAM-depleted preparations, mAb 92OE was subsequently
used to both immunoprecipitate and immunoblot the mAb 13-OE-depleted (lane a) and normal mouse sera-immunoabsorbed
control olfactory bulb preparations (lane b). Similarly, the 161-depleted preparations (lane c) as well as the normal rabbit sera-immunoabsorbed
preparations (lane d) were also both immunoprecipitated and immunoblotted with mAb 9-OE. In both controls (lanes b and d),
mAb 9-OE bound to the 205-kD N-CAM subset. In the N-CAM-depleted preparations (lanes a and c), mAb 9-OE continued to bind,
albeit very weakly, to a 210-kD band.
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Figure 7. Affinity-purified SBA binding molecule from olfactory
bulb. Lanes a-g, respectively, were reacted with ~25I-SBA, mAb
13-OE, mAb 5-OE, mAb 9-OE, 161, anti-HNK-1, and rabbit polyclonal
anti-Ll. Lane a is an autoradiograph and appears darker
against the white blotting membranes used in the other lanes due
to photographic conditions.
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Figure 8. N-CAM was immunoprecipitated from olfactory bulb
with 161 and either deglycosylated with N-glycanase (lanes b, d,
and f) or left intact (lanes a, c, and e) before electrophoresis and
blotting. Blots were subsequently reacted with mAb 13-OE (lanes
a and b), mAb 5-OE (lanes c and d), and mAb 9-OE (lanes e
and f).
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Figure 9. Embryonic day-19.5 rat brain (a) or frog olfactory bulb
(b) membranes were heated at 80°C for 3 min (lanes a, b, e, and
f) or boiled for 30 min (lanes c, d, g, and h) in Laemmli's sample
buffer before electrophoresis. Blots were reacted with 161 (lanes a
and b), mAb 5A5 (lanes c, d, g, and h), or mAb 9-OE (lanes e
and f).
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