Western blot analysis was performed using monoclonal antibodies
(MON-160, MON-161, and MON-162) against human NSP-A and rabbit
antiserum POL-8 against human NSP-C (kindly provided by Wilm J.
M. Van de Ven, University of Leuven, The Netherlands). Among the three
monoclonal antibodies, MON-162 recognized 80% of the human amino acid
residues 338 to 442 (GenBank accession number L10333), which is a
highly conserved region in human and chick
(Fig. 1) . MON-160 and -161
recognized 53% of the amino acid residues 174 to 337 (GenBank
accession number L10333), which were also conserved among animal
species with slightly lower homology. To check for the specificity of
the anti-NSP-A antibodies, Western blot analysis was performed with
MON-162 only and with a mixture of MON-160 and MON-161. These
cross-reacted with an approximately 150-kDa chick retinal protein. No
other bands were observed in the Western blot. POL-8 was raised against
the first 20 unique N-terminal amino acids of human NSP-C, the amino
acid sequence of which was not present in NSP-A and -B
(Fig. 1) . This
sequence (MQATADSTKMDCVWSNWKSQ),
14 however, shares 17
identical amino acids and 2 amino acids similar to the N-terminal amino
acids of chick NSP-C (MQASADSTKMDCLWSNWKCQ). POL-8 was diluted 1:100
and preabsorbed overnight at 4°C with a 10
−4-M
concentration of the chick 20-amino acid peptide (Biologica). This
complex was then used for incubation with the membranes followed by
secondary antibody detection. POL-8 cross-reacted with a 23-kDa chick
protein. The 23-kDa band was eliminated by preabsorption with the
immunizing peptide synthesis. Therefore, both POL-8 and the monoclonal
antibodies (MON-160, -161, and -162) seemed to be suitable for Western
blot analysis and immunohistochemistry. Their cross-reactivities to
chick NSPs were confirmed by Western blot analysis. That is, the
retina–RPE tissues were sonicated in buffer containing 10 mM Tris-HCl
(pH 7.4), 1% (wt/vol) NP-40, 150 mM NaCl, 1 mM EDTA, 20 μM
leupeptin, and 1 mM phenylmethylsulfonyl fluoride at a concentration of
0.1 g wet wt/ml. Each preparation was boiled with Laemmli’s
solution containing 5% 2-mercaptoethanol and loaded into a 5%
SDS-polyacrylamide gel containing 0.1% SDS. Separated proteins were
transferred electrophoretically onto a polyvinylidine membrane
(Millipore, Bedford, MA). The membranes were soaked for 30 minutes in
phosphate-buffered saline (PBS) containing 5% (wt/vol) skim milk and
0.2% (vol/vol) Tween 20 and then washed with PBS. The membranes were
then incubated in a 1:6 dilution of MON-160, -161, and -162 or 1:100
rabbit antiserum POL-8
17 for 24 hours at 4°C. This was
followed by incubation for 2 hours at 4°C with the 1:1000 secondary
anti-mouse IgG horseradish peroxidase (HRP)–linked antibodies
(Amersham) for MON-160,-161, and -162 or 1:1000 anti-rabbit IgG
HRP-linked antibodies (Amersham) for POL-8. For the detection of
immobilized specific antigens conjugated to HRP-labeled antibodies, a
Western blot detection system (ECL Plus; Amersham) was used. The
filters were exposed to x-ray films (Hyperfilm MP; Amersham)
for 16 to 48 hours. Images were digitized and processed as described
earlier.