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. 2013 May 13;288(26):19260–19268. doi: 10.1074/jbc.M112.445270

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Cpc2 is dispensable for ribosomal binding and dimerization of Gcn2. A, schematic diagram of distribution of ribosomes after fractionation using sucrose gradient centrifugation. The predicted absorbance curve at 260 nm is shown along the gradient. B and C, immunoblots of gradient fractions to detect FLAG-Gcn2 and FLAG-rpS3. Whole cell extracts were prepared from Flag:gcn2 rpS3:Flag (YT3650) (B) or Flag:gcn2 rpS3:Flag cpc2Δ (YT3658) (C) cells, and both Gcn2 and rpS3 were detected using anti-FLAG antibody. D, co-immunoprecipitation (IP) assay of Gcn2 dimerization from Flag:gcn2/gcn2⁺ diploid (YT4376), myc:gcn2/gcn2⁺ diploid (YT4281), Flag:gcn2/myc:gcn2 diploid (YT4279), and Flag:gcn2/myc:gcn2 cpc2Δ/cpc2Δ diploid (YT4280) cells treated with 10 mm 3AT 15 min (+) or not (−). Immunoblots were performed using anti-FLAG and anti-Myc antibodies.