Figure 1.

MV151 is a robust tool for detecting β-subunit activity in purified proteasomes and proteasomes from mouse brain and liver tissue.A, structure of the human proteasome (PDB: 5GJR). Modified from Huang et al. (46) and adapted from PyMOL. B, chemical structure of the activity-based probe MV151. Reaction with the N-terminal threonine (purple) of the catalytically active proteasome β subunits is shown. C, structure of the proteasome β ring with highlighted catalytically active β1 (pink), β2 (gold), and β5 (purple) subunits (PDB: 5GJR). Modified from Huang et al. (46). D, purified human 20S proteasome was incubated with vehicle (DMSO), MV151, or an inactive form of MV151 (MV152). MV151-treated samples were incubated with vehicle 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). E, top, purified human 20S/26S proteasomes or lysates from mouse cortical tissue were incubated with vehicle (DMSO) or MV151. Each cortical sample was also incubated with vehicle (DMSO) or the proteasome inhibitor epoxomicin (Epox). All 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 proteasome core subunits (α1–7, β2, β5), a 19S regulatory particle subunit (Rpt5/S6), actin, or neuronal-specific NR2B. β2 and β5 immunoblots show unmodified subunit (arrow), ∼0.5 kDa epoxomicin-shifted subunit (∗), and ∼1.1 kDa MV151-shifted subunit (∗∗). F, top, Cy3 in-gel visualization of MV151 following treatment of purified human 20S/26S proteasome or lysates from mouse liver tissue as in (E). Bottom, representative immunoblots with indicated antibodies following treatment of samples as in (E). Note the presence of catalytically active inducible β subunit (β5i). See also Fig. S1.