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. 2010 Jul 6;22(7):2370–2383. doi: 10.1105/tpc.109.072520

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© 2010 American Society of Plant Biologists

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Figure 6. — PIFs Facilitate Polyubiquitination of phyB by COP1 E3 Ligase.

(A) Effect of PIFs on PhyB-N ubiquitination by COP1 E3 ligase.

(B) Direct interaction between PIF5 and PhyB enhances phyB ubiquitination by COP1.

(C) In vitro pull-down assay demonstrating that PIF5 promotes PhyB/COP1 interaction.

(D) In vitro pull-down assay showing interaction between PIFs (3, 4, and 5) and COP1. Note that compared with the other proteins, the amount of M-PIF3 used was 5 times less.

(E) In vitro ubiquitination assay of PIF3, 4, and 5 by COP1 E3 ligase.

In (A) and (B), in vitro ubiquitination assays were done with E1 ubiquitin activating enzyme and E2 ubiquitin conjugating enzyme in the presence or absence of GST-COP1 (G-COP1).

In (C) and (D), membrane staining (shown below the immunoblots) after the pull-down assay was used to monitor amounts of bait proteins. Input, purified target proteins used in pull-down assays. Numbers in (A) to (C) indicate the relative amounts of proteins in the reaction, where 1 represents 100 ng M-PhyB-N, M-PIFs, M-mPIF5(E31A/G37A), or M-HFR1. Right panel in (A), PIF3, 4, and 5 were added at 50 ng per reaction.