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. 2015 Mar 10;27(3):649–662. doi: 10.1105/tpc.114.132274

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© 2015 American Society of Plant Biologists. All rights reserved.

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Figure 1. — UIMs of DA1, DAR1, and DAR2 Are Required for Ubiquitin Binding.

(A) Phylogenetic tree of the DA1 family members in Arabidopsis. The phylogenetic tree was constructed using the neighbor-joining method of the MEGA 4.1 program. Values at nodes represent percentages of 1000 bootstrap replicates. Alignments used to generate the phylogeny are presented in Supplemental Data Set 1. The scale bar at bottom represents genetic distance.

(B) Schematic diagram of DA1, DAR1, and DAR2 and their derivatives with different domains. aa, amino acids.

(C) UIMs of DA1 and DAR2 interact with ubiquitin in vitro. GST-DA1-UIMs and GST-DAR2-UIMs were pulled down (PD) by His-ubiquitin immobilized on Ni-NTA agarose and analyzed by immunoblotting (IB) using an anti-GST antibody.

(D) UIMs of DAR1 are required for ubiquitin binding. GST-DAR1, GST-DAR1-UIMs, and DAR1^delUIMs without UIM domains were pulled down by His-ubiquitin immobilized on Ni-NTA agarose and analyzed by immunoblotting using an anti-GST antibody.