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. 2012 Aug 15;23(16):3027–3040. doi: 10.1091/mbc.E12-02-0112

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© 2012 Zhang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — NAC binds to the SA segment directly and in a salt-resistant manner. (A) Site-specific cross-linking to ³⁵S-labeled nascent chains (Supplemental Figure S3D). The photo probe was positioned within the SA segment at residue 39 (Dap2-stop39) or 37 (Dap2-stop37) of Dap2-120 (Supplemental Figure S2). Cross-link products were isolated via immunoprecipitation under denaturing conditions using αSrp54, αEgd2, or αEgd1 as indicated and were subsequently visualized via autoradiography. xl Dap2-Srp54, cross-link product between the nascent chain and Srp54; xl Dap2-Egd2, cross-link product between the nascent chain and Egd2; and xl Dap2-Egd1, cross-link product between the nascent chain and Egd1. (B) Chemical cross-linking (Supplemental Figure S3) of RNCs carrying radiolabeled Dap2Δ-62. RNCs were generated in either a wild-type or a Δsrp54 translation extract as indicated. Analysis of the cross-link products was as described in A using αSrp54 and αEgd2. The total represents 33% of the amount added to immunoprecipitation reactions. Samples of cross-link experiments were analyzed via autoradiography. The cross-link between Dap2Δ-62 and Srp54 is labeled with a red asterisk; the cross-link between Dap2Δ-62 and Egd2 is labeled with a green asterisk. (C) RNCs carrying FLAG-tagged (FLAG +) nascent chains as indicated were generated in a wild-type or Δsrp54 translation extract. FLAG–nascent chain pull downs were performed with RNCs isolated under low-salt (L) or high-salt (H) conditions (see Materials and Methods). The isolated material was then analyzed for the presence of SRP (Srp54), NAC (Egd2), and RNCs (Rpl4) via immunoblotting. (D) Dap2-120-RNCs were generated in a translation extract derived from the Δegd1Δegd2 strain. After inhibition of translation, purified NAC was added as described in Figure 4. Subsequently, RNCs were isolated under low-salt (L) or high-salt (H) conditions, and the isolated material was analyzed as in C. (E) RNCs carrying FLAG-tagged (FLAG +) or untagged (FLAG –) 62-residue nascent OM45 (Supplemental Figure S2) were generated in wild-type translation extract. FLAG–nascent chain pull downs were preformed with low-salt-treated (L) or high-salt-treated (H) RNCs and were analyzed as described.