Figure 2.

Epoxomicin in a concentration of 100 nM is sufficient to inhibit proteasome activity. A) Flow cytometric analysis of HeLa and SH-SY5Y cell lines stably expressing Ub^G76V-GFP, a proteasome reporter construct, showed that at a concentration of 100 nM epoxomicin, 95% of all cells accumulated the reporter, indicating a strong inhibition of proteasome activity (n=3 independent experiments). Data are presented as means ± se. B, C) U343 cells transfected with a proteasome reporter construct Ub^G76V-GFP. GFP was degraded without proteasome inhibition (B). After overnight treatment with 100 nM epoxomicin (epox), GFP accumulated in the cells (C). D, E) Fluorescent proteasome activity probe showed fluorescence in all U343 cells treated with DMSO (D) in contrast to U343 cells treated with 100 nM epoxomicin, which hardly showed fluorescence (E). This means that proteasome activity is strongly decreased after treatment with 100 nM epoxomicin. Scale bars = 100 μm (B, C); 50 μm (D, E).