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. 2017 Jan 15;31(2):197–208. doi: 10.1101/gad.292235.116

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© 2017 Dong et al.; Published by Cold Spring Harbor Laboratory Press

This article, published in Genes & Development, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.

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Figure 6. — Functional analyses of DA1 activity in vitro and in vivo. (A) In vitro DA1 cleavage assays using BB-HIS as a substrate for ubiquitylated Flag-DA1 and mutant versions. The top panel shows the reaction at 0 h, and the middle panel shows the reactions at 4 h. The red arrow indicates cleaved BB-HIS. The loading and ubiquitylation status of input HA-DA1 and mutant versions is shown. Quantitative scans showed that BB-HIS was cleaved to a greater extent by wild-type DA1-HIS than by HA-DA1(R358K). HA-DA1(C274Y) and HA-DA1(UIM1+2) did not cleave BB-HIS. (B) In vivo DA1 cleavage reactions using BB-Flag and HA-tagged DA1 and mutant versions. The BB-Flag cleavage product is indicated by an arrow. Quantitative scans showed reduced levels of BB cleavage by HA-DA1(R358K), and HA-DA1(C274Y) did not cleave BB-Flag. HA-DA1(4K-4R) had normal levels of peptidase activity. The loading of HA-DA and its mutant versions is shown in the bottom immunoblot.