This allows a search for relatively rare injection

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This allows a search for relatively rare injection sites, which are at points of connection between the inner and outer membranes. Reference: Goldberg E (1983) Recognition, attachment, injection. In Bacte- riophage T4 (CK Mathews, EM Kutter, G Mosig, PB Berget eds.), pp 32–39. Washington, DC: American Society for Microbiology. DATA HANDLING 19–20 A. Immunoprecipitation of proteins from cells transfected with the full-length construct gave three radiolabeled bands (lane 1, Figure 19–4A). One of these must be the cadherin, itself, and the other two are proteins with which the cadherin interacts. The band corresponding to the cadherin can be identi- fied because it is present in every lane, and it changes position, as expected since each construct expresses a protein of different length; it is the slowest migrating band (top band) in lanes 1, 3, 4, and 5 and the only band in lanes 2 and 6. On SDS polyacrylamide gels, smaller proteins migrate faster than larger ones; thus cadherins with larger deletions are found at lower positions on the gel. The two bands of constant size in lanes 1, 3, 4, and 5 must be the proteins that the cadherin binds. Lane 7 is blank, confirming the specificity of the antibody, and proving that the labeled bands truly represent cadherin binding partners. Incidentally, one of the proteins bound by this cadherin is b -catenin, a central player in the two-way interaction between adhesion and signaling. B. These experiments define the C-terminal 72 amino acid residues of cadherin as the maximum stretch of amino acids required to bind the other proteins. Lane 2 in Figure 19–4A shows that the terminal 37 residues are required since no proteins were bound when that construct was expressed in cells. As shown in lane 5, an internal deletion of 70 amino acid residues did not alter binding, whereas deletion of a further 35 amino acids residues toward the C-terminus (lane 6) eliminated binding. Because the endpoints of the deletions in D C5 and D C10 coincide, the 35- and 37-residue segments form a contiguous 72- amino acid stretch that contains the domain (or domains) that is critical for binding of the other proteins. C. To check whether this domain is sufficient for binding, you might fuse it by cloning to the C-terminus of a heterologous membrane-spanning protein. If this segment of protein is all that is needed for binding, immunoprecipita- tion of the fusion protein should bring down the same two proteins. The researchers who did this study attached the cadherin domain to a class I major histocompatibility antigen, H2-K, and found that antibodies against the extracellular domain of the MHC protein–cadherin fusion also precipi- tated the same proteins. Reference: Ozawa M, Ringwald M & Kemler R (1990) Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule. Proc. Natl Acad. Sci. U.S.A. 87, 4246–4250.
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