FIGURE 5.
Ecm29 impairs the opening of the CP gate in α6K62A proteasomes. A, purified proteasomes (1 μg) were analyzed by 4–12% SDS-PAGE and immunoblotting (IB) with indicated antibodies. Proteasomes were purified with protein A tag appended to a lid subunit, Rpn11 (43), to obtain CP species that had been incorporated into proteasome holoenzymes. Rpn8, a lid subunit, was used as a loading control. B, purified proteasomes (4 μg) from A were resolved by 3.5% native PAGE. The native gel was incubated with LLVY-AMC in the absence of SDS (top). Following photography of the gel, the same gel was further incubated with LLVY-AMC in the presence of 0.02% SDS (middle panel). The native gel was then stained with Coomassie Blue.