Fig. 5: Covalent inhibitors inhibit substrate polyubiquitination but not monoubiquitination. | Communications Chemistry

Fig. 5: Covalent inhibitors inhibit substrate polyubiquitination but not monoubiquitination.

From: Structure-based design of potent and selective inhibitors of the HECT ligase NEDD4

Fig. 5: Covalent inhibitors inhibit substrate polyubiquitination but not monoubiquitination.The alternative text for this image may have been generated using AI.

A Substrate ubiquitination assay with the indicated E3s treated or not with compound 15 (10 µM). The complete reaction was loaded onto a gel. IB as indicated. Arrowhead indicates the unmodified WBP2 protein. B Same as in A, using GST-γENAC as substrate. At the end of the reaction, substrate was separated by centrifugation and loaded onto a gel. IB as indicated. Bottom, Coomassie staining showing comparable loading of proteins. C Same as in A, using GST-Eps15 as substrate. Top panel, experiment performed with NEDD4, bottom panel with NEDD4-Like. Coomassie staining showing comparable loading of proteins are reported. D B82L cells were serum starved overnight, treated with compound 15 (1 µM) for 4 h and then stimulated with EGF (100 ng/ml) for the indicated time points. Lysates were analyzed by IB with the indicated antibodies.

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