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. 2012 Jan 24;287(12):9269–9279. doi: 10.1074/jbc.M111.316208

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© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — ATP hydrolysis by Rpt subunits is not essential for proteasome assembly. A, comparable incorporation of wild-type and Walker B mutant Rpt subunits into proteasome. HeLa cells were transiently transfected with the expression constructs of FLAG-tagged wild-type or Walker B mutant Rpt subunits as indicated. Aliquots of FLAG-Rpt samples purified in the presence of 5 mm ATP were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. B and C, immunoblotting (B) and SDS-PAGE (C) analyses of affinity-purified FLAG-tagged wild-type or Walker B mutant Rpt3. HeLa cell lines that conditionally expressed FLAG-tagged wild-type or Walker B mutant Rpt3 were established, and the proteasome was affinity purified as described under “Experimental Procedures.” Ten microliters each of Rpt3 samples purified in the presence of 5 mm ATP was analyzed by immunoblotting with indicated antibodies and by SDS-PAGE followed by Coomassie staining. D and E, immunoblotting (D) and SDS-PAGE (E) analyses of affinity-purified FLAG-tagged wild-type or Walker B mutant Rpt6.