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S.-P. Huang, B. M. Brown, C. M. Craft; N-Ethylmaleimide Acts as a Functional Partner for Visual Arrestin1 in the Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5430.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the molecular and cellular mechanisms of the interaction between Arr1 and NSF in maintaining the normal synaptic function in the mouse photoreceptors.
The interaction of Arr1 and NSF was first identified using co-immunoprecipitation (IP) assays, followed by mass spectrometry (MS) analysis, and verified by immunohistochemical methods. The transcription levels of NSF were analyzed by quantitative RT-PCR. The region in NSF that interacts with Arr1 was defined by constructed NSF-truncated domain segments (amino acids residues 1-744, 1-205, 206-744, 1-477 and 1-206^478-744) and analyzed using gel overlay assay.
Co-IP assays of mouse retinal homogenates using Arr1-specific antibody (D9F2) identified NSF by MS analysis and supports their potential interaction. Moreover, protein-protein interactions between Arr1 and NSF were greater in the retina isolated in dark conditions. Immunohistochemical analysis reveals NSF and Arr1 are co-localized in the photoreceptor synapse of the dark-adapted retinas with more intense staining compared to the light-adapted retina. NSF mRNA expression level is significantly increased in dark-adapted compared with light-adapted retinas, while NSF transcription level is decreased in the Arr1-/- retinas compared with control retinas in dark conditions. Gel overlay assay of recombinant Arr1 protein with serial NSF truncated proteins showed that Arr1 binds to the junction of N-terminal (N) and first ATPase (D1) domain of NSF.
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