Xenbase Image ID: 144595

Figure 2. BiFC assay based on Venus, a highly fluorescent YFP (yellow fluorescent protein) variant (Nagai et al., 2002), allows detection of additional homotypic interactions by widefield epifluorescence microscopy. A, Coexpression of complimentary pairs of Venus-based fusion proteins generated modest BiFC signals for both GARP2 and rhodopsin, consistent with reports that these proteins can weakly self-associate. IHC analysis of the individual partners (HA, red; MYC, fuchsia) shows them to be overlapping with, but more broadly distributed than the BiFC signals (green). Self-assembly of P/rds produced robust BiFC under similar conditions (data not shown). B, BiFC assay of proposed heterotypic interactions (Poetsch et al., 2001) between GARP2 and P/rds. Coexpression of Va-HA-GARP2 and P/rds-MYC-Vb partners produced a moderate BiFC signal. IHC analysis of the partners shows them to be overlapping with but more broadly distributed than the BiFC signal. In contrast, coexpression of Va-HA-GARP2 with rhodopsin (Rho-MYC-Vb) did not produce significant BiFC, despite robust expression of each partner. IHC epitope tag detection was specific, since reactivity could be blocked by synthetic peptide preincubation. All combinations of complementary GARP2-P/rds partners (such as GARP2-HA-Va and P/rds-MYC-Vb shown) were found to produce similar levels of BiFC. 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

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