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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Cancer Res. 2015 Nov 5;75(23):5130–5142. doi: 10.1158/0008-5472.CAN-15-1544

Figure 3. Naturally occurring ITCs inhibit USP9x and UCH37 in cell lysates and in living cells.

Figure 3

A, BaF3/p210 cells were incubated with PEITC or BITC for 4 h. The cells were harvested, washed, resuspended in lysis buffer and lysed with glass beads. After centrifugation, the clarified supernatant was normalized (0.6 mg/mL), then Cy5-UbVME (250 nM) was added and the mixture incubated at 37 °C for 5 min and analyzed by SDS-PAGE. Ingel fluorescent scan was obtained on Typhoon scanner. Data representative of three independent experiments. Asterisks denote DUB assignments that were verified by immunoblotting. In cases where the bands were not confirmed by immunoblotting, DUBs were identified based on accepted MW. B, A BaF3 cell lysate (12 mg/mL) was incubated with PEITC or BITC (250 μM), or with DMSO vehicle (1% final concentration) and then diluted 20-fold into buffer containing HA-UbVS (1.5 μM). Aliquots were removed at the indicated times resolved by SDS-PAGE and immunoblotted with anti-HA. `-' indicates no compound. C, A BaF3 cell lysate was incubated with PEITC or BITC, treated with HA-UbVS, resolved on a 6% polyacrylamide gel and immunoblotted with anti-USP9x and anti-USP7. `-' indicates vehicle control. D, A BaF3 cell lysate (1.5 mg/mL) was incubated with PEITC, BITC, or SFN (50 μM) or with G5 or WP1130 (10 μM) for 10 min prior to addition of HA-UbVME (1.5 μM). After 20 min aliquots were analyzed by immunoblotting with anti-UCH37 anti-body. `Ratio' represents UCH37-UbVME conjugate per total UCH37. Data representative of two independent experiments. `-' indicates no compound, vehicle only. E, A BaF3 cell lysate (1.5 mg/mL) was incubated with PEITC or BITC, treated with HA-UbVS (1.5 μM) and analyzed by immunoblotting with anti-USP24. Data are representative of two independent experiments. F, 19S regulatory particles (19S RP, 30 nM) were treated with PEITC or BITC for 30 min at which time Cy5-UbVME (300 nM) was added. After 20 min, aliquots were quenched in loading buffer and resolved by SDS-PAGE. Ingel scans were obtained on a GE Typhoon scanner.