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. 2011 Jul 6;286(35):30274–30283. doi: 10.1074/jbc.M111.240309

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — Mapping of the proteasome binding region of P97. A, full-length p97 or truncated varients were expressed as FLAG-tagged fusion proteins in HEK 293T cells treated with MG132. Expression of FLAG-tagged p97 proteins was detected by immunoblotting before (input left panel) and after IP of PSMA1 (right panel). Note the importance of D1 and D2 domains for P97-proteasome complex formation. B, wild type p97 or truncated proteins were expressed in TNT reticulocyte expression system and subjected to glycerol gradient centrifugation. The collected fractions were resolved on SDS-PAGE and exposed to autoradiography. The hexameric form of P97 proteins is indicated. Flag P97 linker +D2 (461–806) and Flag P97 ΔD2 (1–480) fail to form hexamers.