Since canine ANGPTL7 is 97% identical with the human protein, we performed immunohistochemistry on canine tissue and Western blot analysis of canine AH, using the anti-human ANGPTL7 antibody. The ANGPTL7 staining pattern for canine eyes
(Figs. 7A 7B 7C)was very similar to that in human eyes
(Fig 4) . As with human tissue, ANGPTL7 staining (brown) was strong in the limbal area of the cornea, with gradual reduction in staining intensity toward the central cornea
(Fig. 7A) . Also similar to humans, ANGPTL7 staining was found throughout the sclera, but not in the corneal epithelium
(Fig. 7A) , retina, or choroid
(Fig. 7B) . Within the canine optic nerve, ANGPTL7 immunoreactivity was found within the cribriform plates, similar to the staining pattern in the human optic nerve
(Fig. 4E) . No immunoreactivity was observed with negative control IgG substituted for the anti-ANGPTL7 antibody (data not shown). In Western blot analysis of canine AH, the antibody against human ANGPTL7 recognized canine ANGPTL7 under reducing conditions as immunoreactive bands of ∼50 kDa, similar to human AH (
Fig. 7D , cf.
Fig. 6A ) and corresponding to monomers of canine ANGPTL7. However, the bands were faint and diffuse under reducing conditions and difficult to detect. Under nonreducing conditions, prominent well-defined bands of ∼250 kDa and fainter bands of ∼210 kDa were detected
(Fig. 7E) , indicative of multimers of ANGPTL7. This is expected, since ANGPTL7 and other ANGPTL family members form disulfide-linked multimers which, under nonreducing conditions, display several high molecular weight bands corresponding to various degrees of oligomerization.
17 24 26 The shift in molecular weight is similar to human ANGPTL7 immunoreactivity in ANGPTL7-transfected TM cells, which showed 48-kDa bands in the presence of reducing reagent and a ∼250-kDa band without it
(Fig. 1C) .