Inhibition of the proteasome by carfilzomib. (A) Structures of epoxomicin (top) and carfilzomib (bottom) are shown. (B) Quantitative representation of the in vitro inhibition of the 20S proteasome catalytic activities in ANBL-6 cellular lysates (10 μg per reaction) in the absence or presence of carfilzomib with fluorogenic peptide substrates for the proteasomal ChT-L, PGPH, and T-L activity, as indicated, followed by the measurement of free AMC groups is shown. (C) In cellulo measurement using ELISA techniques of the 20S proteasome subunit targets of carfilzomib in cell extracts from ANBL-6 cells pulse treated for 1 hour with carfilzomib is shown. Error bars in panels B and C are SD. (D) Competitive binding experiment in ANBL-6 cells between carfilzomib (5-hour pretreatment) followed by VS-L3-AHx3-danysl (2 hours) determined the in cellulo specificity of carfilzomib to individual proteasome catalytic subunits. (E,F) Shown is Western blot analysis of the accumulation of ubiquitinated (Ub) substrates (E) and proapoptotic Bax (F) after 1-hour pulse exposure to carfilzomib (100 nM) in RPMI 8226 cells, followed by 24- and 48-hour recovery times. Actin was used as a loading control. Veh indicates vehicle.
