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. Author manuscript; available in PMC: 2020 Jan 29.
Published in final edited form as: Methods Enzymol. 2019 Jan 3;619:145–159. doi: 10.1016/bs.mie.2018.11.006

Figure 2.

Figure 2.

A. Conformation-dependent crosslinking of Rpn7 and Rpt2. Crosslinking requires both engineered cysteines and is regulated by nucleotide. Crosslinking was induced in the presence of 2 mM of the indicated nucleotide. For the last lane, the WCE was pre-incubated with DTT to reduce disulfide crosslinks prior to loading. B. Bortezomib (BTZ) treatment does not influence Rpn7-Rpt2 crosslinking. Extracts from DMSO or BTZ (100 μM) treated cells were crosslinked as in (A) in the presence of 2 mM ATP or AMP-PNP as shown. Asterisk, nonspecific cross-reacting band occasionally seen with V5 antibody. C. Measurement of proteasomal peptidase activity in extracts from (B). Cell extract (20 μg) from DMSO- or BTZ-treated cells was incubated with 50 μM suc-LLVY-AMC for 60 minutes at 30°C and fluorescence from liberated AMC was measured. The rate of fluorescence liberation is shown. AFU, arbitrary fluorescence units. Error bars indicate standard deviations (N = 5). Panel A is modified from (Eisele et al., 2018) with permission from Elsevier.