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. Author manuscript; available in PMC: 2008 Jun 18.
Published in final edited form as: J Neurochem. 2007 Aug 6;103(3):1053–1062. doi: 10.1111/j.1471-4159.2007.04842.x

Fig 1.

Fig 1

Similar localization of endogenous cone arrestin in cone photoreceptors and HEK293 cells. (a) Endogenous cone arrestin is predominantly cytoplasmic in photoreceptor cells. Cone arrestin in salamander retina was visualized with F4C1 antibody (green). Lower panel shows overlay of cone arrestin immunostaining (green) and nuclear staining (4′,6-diaminido-2-phenylindole, blue). OS, outer segments; ONL, outer nuclear layer (photoreceptor nuclei); INL, inner nuclear layer. (b) The distribution of cone arrestin in HEK293A cells. Flag-and green fluorescent protein (GFP) -tagged cone arrestin was visualized with M2 anti-Flag antibody (red), and by native GFP fluorescence (green), respectively. Untagged and flag-tagged cone arrestin localizes almost exclusively in the cytoplasm, similar to endogenous cone arrestin in photoreceptors. GFP-tagged cone arrestin (which is ~20 kDa bigger) is also predominantly cytoplasmic, with a slight increase of the nuclear content. Exportin1 inhibitor leptomycin B (LMB) had little effect on the distribution of untagged and Flag-tagged cone arrestin, whereas the distribution of GFP-tagged protein changed dramatically from predominantly cytoplasmic to almost exclusively nuclear. An engineered nuclear export signal (NES) had minor effect on arrestin distribution with or without LMB treatment. Untagged, Flag- and GFP-tagged cone arrestin were expressed at comparable levels of 71 ± 9, 50 ± 15, and 66 ± 6 pmol/mg protein, respectively, as measured by quantitative western blot with F4C1 antibody with calibration curve with purified recombinant cone arrestin. (c) C > N: The proportion of cells showing stronger fluorescent arrestin signal in the cytoplasm than in the nucleus. Mean ± SD of three experiments are shown. ****p < 0.0001 (green), indicates the significance of LMB effect. Cone and Cone-F, untagged and Flag-tagged cone arrestin; Cone-NES and Cone-NES-F, untagged and Flag-tagged cone arrestin with an engineered NES in the C-tail; Cone-GFP, GFP-tagged cone arrestin; Cone-NES-GFP, GFP-tagged cone arrestin with an engineered NES.