Design of ubiquitination-induced fluorescence complementation (UiFC) assay.

<p>(A) Schematic representation of UiFC-N and UiFC-C and the proposed mechanism of UiFC for detecting polyubiquitin chain. (B) UiFC-N and UiFC-C interact with K11, K48, and K63 chains. Purified 6His-UiFC-N and 6His-UiFC-C were incubated with K11, K48, or K63 chains. Following the incubation immunoprecipitation was carried out with anti-ubiquitin antibody followed by immunoblotting for venus. Control IP: anti-HA antibody was used instead of anti-ubiquitin. Input: 10% of total UiFC-N and UiFC-C or polyubiquitin chains. Aristerisk indicates UiFC-N. Arrowhead indicates UiFC-C. (C) Spontaneous reconstitution of the N and C-terminal fragment of venus in UiFC-N and UiFC-C <i>in vitro</i>. Increasing amounts of 6His-UiFC-N and 6His-UiFC-C were incubated in 20 µl Tris buffer and venus fluorescence was measured every 10 minutes for up to 270 minutes at 37 °C as described in Experimental Procedures. The mean +/- SD of venus fluorescence intensities from triplicate reactions at each time point was plotted. (D) K48 ubiquitin chain but not mono-ubiquitin induces increases in venus fluorescence. UiFC-N and UiFC-C (0.86 µM) were incubated with the reaction mix for K48 chain synthesis containing 2 µM ubiquitin in the presence or absence of ATP for 60 minutes. Control: no K48 chains, DUB: 6His-EBV-DUB. The graph shows the mean +/- SD of UiFC fluorescence intensities from triplicate reactions (left). (E) The reaction mixes used in D were analyzed by immunoblotting for ubiquitin after the measurement of venus fluorescence. Lower panel: the added 6His-EBV-DUB revealed by Ponceau staining.