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Figure 7. Immunoprecipitation analyses demonstrate a segregated assembly of transgenic proteins. Reactions were assayed by Western blotting using the indicated antibodies; blots represent quantitative comparison of: detergent lysate (LYS), unbound supernatant (UB), and eluted (ELU) fractions. A, A novel monoclonal antibody, Mab 1G9, was developed to the C terminus of xP/rds (xrds38) and was used to immunoprecipitate (IP) the protein from Triton X-100 tadpole eye extracts under nonreducing conditions. The endogenous protein (most commonly observed as a doublet) could be effectively depleted from lysates (UB) and then recovered following SDS (ELU1), but not acid (ELU2) elution of beads. Importantly, Mab 1G9 showed no cross-reactivity with bP/rds. We found that bP/rds (detected with ortholog-specific MabC6) remained in lysates from which xP/rds was efficiently removed. B, Left, Immunoprecipitation of endogenous xP/rds from transgenic tadpole eyes coexpressing complementary Venus fragments fused to the following: (1) bP/rds lacking its C terminus (Va-HA-bP/rdsΔC); and (2) full-length bP/rds (Vb-MYC-bP/rds). The vast majority of each transgenic fusion protein (detected with polyclonal antibody Pab290) failed to coprecipitate. Right, Immunoprecipitation of endogenous xP/rds from transgenic tadpole eyes expressing bP/rds fused to full-length GFP protein at its C terminus (bP/rds-C150S-GFP). Little or no coprecipitation with endogenous xP/rds was observed. C, Reciprocal immunoprecipitation performed with an anti-GFP matrix confirms segregated assembly. Left, Immunoprecipitation of a xP/rds-GFP fusion protein from transgenic tadpole eyes. Transgenically expressed fusion protein (detected with polyclonal antibody Pab290) was efficiently immunoprecipitated; however, neither endogenous xP/rds (detected with Mab 1G9) or rhodopsin (K62-82) was coprecipitated. Right, What appeared to be a minor amount of coprecipitating xP/rds was in fact, a cross-reacting protein also present in WT tadpole eye lysates. D, Assembly of a P/rds C-terminal deletion mutant. A reciprocal immunoprecipitation performed using the lysate detailed in B above (coexpressed Va-HA-bP/rdsΔC and Vb-MYC-bP/rds). BiFC complexes were collected using an anti-GFP/Venus matrix and blotted for individual fusion protein partners. Although transgenic proteins coprecipitate with each other, endogenous P/rds does not. MabC6 binds the C terminus of bP/rds and therefore is specific to the full-length protein. 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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