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ies. Immunohistochemical staining of the salamander retina demonstrates stronger staining
for HCNl antibodies than HCN3 for rod and cone photoreceptors (figure 3.2). Anti-HCNl
antibodies strongly label the rod and cone inner segments, with some binding in the outer
segments as well. This finding agrees with our electrophysiology data that indicates that the
HCN1 isoform is dominant in these cells, however, we did not observe any HCN currents
from the outer segments. HCN3 binding appears to be relatively nonspecific, with some
binding in the photoreceptors.
To test the specificity of the antibodies used, a western blot was performed on salaman-
der retina and mouse brain. For HCNl in mouse brain (figure 3.2 Al), a single band ap-
pears in the 100-110 kDa range, which is consistent with the predicted molecular weight
for mouse HCNl (102 kDa). For salamander, a single band appears near 60 kDa (figure 3.2
A1 ), which is consistent with findings of other researchers from the rat retina [83 ]. Although
the observed molecular weight is different than the mouse, the appearance of single specific
band indicates that the HCNl antibody does indicate specific binding in the salamander. A
BLASTp search on the Xenopus tropicalis HCNl protein sequence [20] predicts binding of
the HCNl antibody to amphibian HCNl, with homology in 15 of 19 amino acids between
the antibody epitope and Xenopus HCNl. The 60 kDa band in retina has been observed by
other researchers also using N-terminal HCNl antibodies in rat retina [83]. It is possible
that the 60 kDa protein is a truncated form of HCNl. Western blot with the HCN3 antibody
(figure 3.2 Bl) demonstrated distinct bands near 50 kDa in both mouse brain and salaman-
der retina. These bands may both represent protein fragments, as the predicted mass for