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. Author manuscript; available in PMC: 2010 Dec 24.
Published in final edited form as: Chem Biol. 2009 Dec 24;16(12):1278–1289. doi: 10.1016/j.chembiol.2009.11.015

Figure 3. NC-005 derived active site probe and its inactive diastereomer demonstrate that NC-005 is proteasome specific.

Figure 3

A RPMI-8226 cells were treated with az-NC-005 active site probe. Activities were measured (values are averages ± S.E. of two experiments) and, in parallel, extracts of these cells were treated with biotinylated phosphane (Fig. 4G) and analyzed by Western blotting. Az-NC-005 reacting polypeptides were visualized by staining with fluorescently labeled streptavidin and identified based on the well-characterized pattern of migration of proteasome’s catalytic subunits (Altun et al., 2005). Endogeneously biotinylated protein of ~70 kDa served as a loading control. B. RPMI-8226 cells were treated with 1.6 µM NC-005 and (S)-NC-005 for 6 h. Proteasome activities were measured immediately after the treatment using peptides substrates (Table); cell viability was measured after subsequent 42 h cultivation in inhibitor-free media.