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T. K. Lee, S. Iragavarapu, G. Gaidosh, R. K. Lee, S. K. Bhattacharya; Localized Expression of Disco-Interacting Protein 2 Homolog A (Dip2a) in Human Eyes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):786.
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Proteomic analysis of human aqueous humor (AH) and lens capsule (LC) unexpectedly revealed the presence of Disco-interacting protein (drosophila) homolog 2 (Dip2a) protein. This study aims to confirm the expression of Dip2a in the human eye and to determine which structures in the eye express Dip2a.
: AH and LC samples were collected during cataract surgery and promptly stored at -80ºC for analysis. Frozen samples of 8-10 LC were pooled, homogenized in extraction buffer (125mM Tris-HCl, 1% SDS and 100mM NaCl ), centrifuged at 10,000xg for 10 minutes, and the supernatant was collected. Soluble protein was quantified by Bradford assay. AH and LC soluble proteins were fractionated on SDS-PAGE and Dip2a protein expression was detected with Western blotting using custom-generated affinity purified chicken antibodies against Dip2a peptide sequences. Dip2a expression was also detected by immunostaining of human globe sections.
Dip2a was detected by Western blot analysis, in agreement with presence of Dip2a in human AH and LC detected by proteomic MS/MS mass spectrometry analysis. Immunofluorescence of human globe sections demonstrated the expression of Dip2a in the human ciliary body and in the retina plexiform layers.
Dip2a, a drosophila homolog gene with poorly understood function, encodes a protein with homology to lipid metabolism, transcriptional regulation, and axonal guidance genes. In agreement with Cepko’s SAGE library, Dip2a is expressed in the retina (especially in the plexiform layers of the retina) and in the ciliary body based upon immunofluorescence. In addition, our proteomic and Western blot data demonstrated that Dip2a is also expressed in AH and LC. This is the first report of Dip2a expression in the human eye.
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