Xenbase Image ID: 144594

Figure 1. A GFP-based BiFC assay reports photoreceptor protein interactions in cultured cells. A, The approach uses fusions of nonfluorescent N- and C-terminal fragments of GFP (or YFP, yellow fluorescent protein) to partners of interest. Interaction of the partners (X, Y) brings the complementary fragments into proximity, promoting fluorophore maturation. B, GFP fragment fusion protein variants encoding photoreceptor proteins—P/rds, rhodopsin, and GARP2. C, Western blot analysis of fusion protein expression in transiently transfected HEK293 cells. The results demonstrate that all constructs can be expressed and that the epitope tag antibodies react specifically. D, Coexpression of complementary (GFPa and GFPb) fragments fused to either the N or C terminus of P/rds produced relatively modest BiFC signals (green) imaged by widefield epifluorescence microscopy. IHC analysis of the individual partners (HA, red; MYC, fuchsia) shows them to be overlapping with, but more broadly distributed than the BiFC signals. A similar assay applied to GARP2 and rhodopsin produced no significant signals, although previous reports suggest that each may engage in weak self-association (Batra-Safferling et al., 2006; Fotiadis et al., 2004). In sum, these results suggest that a BiFC assay based on GFP fragments is sufficient to detect strong, but not weak, interactions. Image published in: Ritter LM et al. (2011) Copyright © 2011. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

Return to previous page