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. 2011 Aug 30;286(42):36652–36666. doi: 10.1074/jbc.M111.285924

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

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FIGURE 6. — Enhanced Ecm29 binding to proteasomes in RP assembly mutants, rpt4-Δ1 and rpt6-Δ1. A, whole cell extracts (75 μg) were analyzed using 3.5% native gels and LLVY-AMC assays in the presence or absence of 0.02% SDS. In parallel, 20 μg of whole cell extracts were subjected to SDS-PAGE and immunoblotting (IB) for Ecm29. eIF5A is a loading control. B, phenotypic analysis of rpt-Δ1 mutants and hsm3Δ rpn14Δ nas6Δ RP chaperone mutants. 5-fold serial dilutions of yeast strains were spotted on YPD plates or in the presence of 0.4 μg of 4-NQO or 0.035% MMS. The plates were incubated for 2–3 days at 30 °C or otherwise as indicated. C, SDS-PAGE and immunoblotting of whole cell extracts (20 μg). eIF5A is a loading control. D, real time PCR analysis of mRNA levels of the indicated genes. E, native PAGE and LLVY-AMC assay as in A with whole cell extracts. F, immunoblotting of the native gels from E. Rpn8, a lid subunit.