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. 2016 May 9;171(3):1808–1820. doi: 10.1104/pp.16.00619

Figure 2.

Figure 2.

Biochemical characterization of the Vx3K0 and Vx3NB sensors. A, Scheme representing the principle of K63 polyubiquitinated protein purification using Vx3K0 (left) and the nonbinding Vx3NB as a control (right). B, In vivo characterization of purified proteins using sensor-based immunoprecipitation of proteins carrying K63 polyubiquitin chains. Immunoprecipitation (IP) was performed using anti-GFP antibodies on RIPA buffer-solubilized protein extracts from wild-type (WT) and monoinsertional homozygous plants expressing free GFP, Vx3K0-HA-GFP, and Vx3NB-HA-GFP and subjected to immunoblotting with anti-GFP, anti-ubiquitin P4D1, anti-K63 polyubiquitin Apu3, and anti-K48 polyubiquitin Apu2 antibodies. The membrane was probed with the respective antibodies and stripped in the following order: anti-GFP, anti-K63 polyubiquitin (Apu3), anti-K48 polyubiquitin (Apu2), and anti-ubiquitin (P4D1) antibodies. The sizes of marker proteins in kD are indicated next to the respective blots. The asterisk indicates a nonspecific band recognized by the anti-GFP antibodies. Note that both Vx3K0 and Vx3NB sensors show partial cleavage, as attested by the presence of a signal migrating at the size of free GFP.