Figure 1.

PdPT is an inhibitor of multiple DUBs. a and b The in situ and in vitro effects of PdPT on proteasome peptidase activity. Lysates were from PdPT or BTZ-treated A549 (A) and NCI-H1299 (B). Cells were treated with the indicated doses of PdPT and then the proteasome chymotrypin-like activity were analyzed. (C) Purified 20S proteasomes were treated with the indicated doses of PdPT and then the chymotrypin-like activity was measured using specific synthetic fluorogenic substrates. BTZ was used as a positive control. Values are expressed as mean ± SD (n = 3). **P < 0.01, compared with each control. d and e The effect of PdPT on DUB activities. Purified 26S proteasomes (D) or A549 cell lysates (E) were exposed to PdPT (1.25, 2.5, and 5.0 μM) and the dynamic DUB activity was measured. NEM was used as a positive control. (F) Abolishment of Ub-VS labeling of proteasomal DUBs by PdPT. Lysates from PdPT (2.5, 25 μM) or b-AP15 (25 μM)-treated A549 and NCI-H1299 cells were incubated with labeled HA-UbVS at 37°C for 30 min. The levels of HA and DUBs were assayed using western blot. (G) A549, NCI-H1299, and A549/DDP cells were treated with indicated PdPT for 6 h. Total ubiquitinated proteins (Ubs.), K48-linked ubiquitinated proteins (K48), and PARP proteins were detected using western blot. Bortezomib(BTZ, 100 nM) and b-AP15 (1 μM) were used as a positive control and CDDP (10 μM) was used as a negative control.