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. Author manuscript; available in PMC: 2020 Jan 15.
Published in final edited form as: Bioorg Med Chem Lett. 2018 Nov 28;29(2):204–211. doi: 10.1016/j.bmcl.2018.11.055

Figure 2. Diarylcarbonates inhibit the decomposition of high molecular weight ubiquitinated proteins (HMW-Ub).

Figure 2.

Lysates were prepared from HEK 293T cells expressing HA-ubiquitin. Samples were incubated at 37 °C and reactions were quenched by the addition of reducing Laemmli buffer. HMW-Ub was assessed by SDS-PAGE and immunoblotting with anti-HA antibody. A. Representative immunoblots measuring the decomposition of HMW-Ub in lysates treated with either the DMSO vehicle, G5 (10 μM) or C4 (500 μM). B. Plot of the decomposition of HMW-Ub in lysates treated with DMSO and C4 (500 μM) (N = 2; error bars denote range). C. The effect of proteasome and DUB inhibitors on the decomposition of HMW-Ub (N=2, average and range are shown). D = DMSO, B = bortezomib (a proteasome inhibitor); N = NSC 632839 (a broad spectrum DUB inhibitor); G5 = G5 isopeptidase inhibitor 1 (a broad spectrum DUB inhibitor); relative to the control (DMSO alone at time = 0). D. Representative decomposition of HMW-Ub after 2 h incubation in the presence of varying concentrations of C4. E. Quantitation of D.