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

FIGURE 3.

FIGURE 3.

Ecm29 promotes premature CP assembly with RP in α5K66A and α6K62A. A, a native gel as in Fig. 1A was used to excise the gel regions containing proteasome holoenzyme species, RP2-CP or RP-CP, into horizontal strips. These strips were subjected to SDS-PAGE and immunoblotting (IB) for CP subunit β2. Pro-β2 is an immature form containing N-terminal propeptide; m-β2 is the mature form. B, whole cell extracts (70 μg) from indicated strains were resolved on 3.5% native gels and incubated with LLVY-AMC in the presence or absence of 0.02% SDS to visualize proteasomes. Following the LLVY-AMC assay, the native gels were subjected to immunoblotting with anti-Ecm29 antibody. C, a native gel as in B was used to excise RP2-CP or RP-CP species as horizontal strips, which were then subjected to SDS-PAGE and immunoblotted for subunit β2. D, the native gels from B were immunoblotted to detect proteasomes and their subassemblies. E, whole cell extracts from B were separated by 4–12% SDS-PAGE and immunoblotted for proteasome subunits. Pgk1 is a loading control. F, 5% native PAGE of whole cell extracts from B. β2, a CP subunit; Rpn5, a lid subunit; Rpt1, a base subunit.