Figure 5.

Human brain tissue contains active 20S proteasome complexes with similar β subunit kinetics to that observed in mice as measured by MV151.A, top, human brain homogenate was incubated with vehicle (DMSO), MV151, or an inactive form of MV151 (MV152). MV151-treated samples were incubated with vehicle (DMSO) or proteasome inhibitor MG132. Samples were applied to SDS-PAGE and imaged with the Cy3 channel to detect MV151 labeling. MV151 bands correspond to individual catalytically active β subunits (arrows). Bottom, MV151 gels were immunoblotted with antibodies to the indicated core proteasome subunits (β2, β5) and actin. β2 and β5 blots show unmodified subunits (arrow) and ∼1.1 kDa MV151-bound shifted subunits (∗∗). B, top, human brain homogenate was incubated with MV151, and samples were collected at indicated time points. Samples were applied to SDS-PAGE and imaged with the Cy3 channel to detect MV151 labeling. Bottom, MV151 gels were immunoblotted with antibodies to the indicated core proteasome subunits (β2, β5) and actin. β2 and β5 blots show unmodified subunits (arrow) and ∼1.1 kDa MV151-bound shifted subunits (∗∗). C, top, human brain homogenate was incubated with MV151 and processed under native conditions. Samples were applied to 4% native gel and imaged with the Cy3 channel to detect MV151 labeling. Bottom, imaged gel was then immunoblotted with antibodies to the indicated core proteasome subunits (α1–7, β5). Cy3 and immunoblot bands correspond to individual proteasome complexes (30S, 26S, 20S) (arrows). Note: we only detect the 20S proteasome in human brain tissue. See also Fig. S5.