Fig 2. ETPs inhibit protein degradation in vitro and in cells.

(A) SOP6 and gliotoxin block the processing of ^K48UbⁿGST–Wbp2 by the proteasome. ^K48UbⁿGST–Wbp2 was incubated with 26S proteasome at 37 °C in the absence or presence of SOP6 or gliotoxin (10 µM each). (B) ETP treatment caused accumulation of high-molecular weight ubiquitin conjugates. HCT116 cells were treated for 3 hours with 10 μM of the indicated ETPs, and the cell lysates were fractionated by SDS–PAGE and immunoblotted with antibodies against ubiquitin. (C) SOP6 and gliotoxin do not inhibit chymotrypsin-like activity of the proteasome. Suc-LLVY-amc (20 μM) was incubated with 15 nM purified human 26S proteasome in the absence or presence of SOP6, gliotoxin or BTZ (20 µM each).