Immunodetection of zona pellucida-like domain protein from salmon samples.

<p><b>A</b>, Coomassie stained SDS-polyacrylamide gel and B, corresponding Western blot of separated crude cupula extracts. Lane 1, untreated sample, lane 2, PNGase F treated sample with faster migration of zona pellucida-like domain protein. <b>C</b>, HE stained inner ear cross section, asterisk marks the cupula and the arrowhead the subcupulary region with sensory and supporting cells. <b>D</b>, immunostaining of inner ear cross section, the arrow probably indicates staining of supporting cells which produce the zona pellucida-like domain protein. This is even more pronounced in <b>E</b>.</p

Visualization of cupula proteins.

<p><b>A</b>, crude extracts from isolated cupulae from salmon, (lane 1) and chicken (lane 2) were separated on a 12% SDS-PAGE under reducing conditions and silver stained. The arrowhead highlights a dominant protein (∼45 kDa) chosen for further analyses. Lane 3, marker proteins. In the 60 kDa range additional yet unidentified protein components are visible. <b>B</b>, deglycosylation of salmon cupula protein extract. Lane 1, cupula extract untreated; lane 2, cupula extract+PNGase F (100 NEB units), lane 3, PNGase F control (500 NEB units). Arrowheads indicate molecular weight shift of the 45 kDa protein due to the <i>N</i>-deglycosylation.</p

Peptide sequences obtained from the 45 kDa gel band from salmon.

<p>Data base searches were performed using Mascot and annotation of the MS/MS spectra was done manually. Amino-acid residues that differ from the published salmon sequence (C0H9B6) are bold. Amino-acid modifications: pyroQ, pyroglutamate, (delta mass: −17); ox, oxidized methionine (delta mass: +16); cam, carbamidomethyl, (delta mass: +57).</p>a<p>peptide with additional amino-acid exchange.</p><p>*peptide with additional modification.</p><p>Peptide sequences obtained from the 45 kDa gel band from salmon.</p

The cupula.

<p><b>A</b>, localization of the cupula in the inner ear. <b>B</b>, dissected cupula from salmon stained with Evans blue.</p