Fig. 1: Fusion of TUBE with E3 increases the efficiency of identification of substrate candidates.

a Work flow for identifying substrates of an E3 ubiquitin ligase. b Construction of an N-terminal FLAG–TUBE-fused probe. The TUBE consists of four UBA domains of the human RAD23A gene with a flexible linker. c Establishment of HEK293 Tet-On 3G cells that inducibly express FLAG–TUBE. Cells were not treated or were treated with doxycycline (1 μg/ml) for 72 h to express FLAG–TUBE. Cell lysates were immunoprecipitated with anti-FLAG M2 agarose. The precipitates were analyzed by immunoblotting with the indicated antibodies. d, e Venn diagrams for identified substrate candidates in Parkin experiments with CCCP (10 μM) treatment for 1 h (d) and TRIM28 experiments (e) in HEK293T cells. To express FLAG–TUBE and each E3 ligase independently, HEK293 Tet-On 3G cells stably expressing each E3 ligase were treated with doxycycline (1 μg/ml) for 72 h to express FLAG–TUBE. HEK293 Tet-On 3G cells that inducibly express FLAG–TUBE alone were used as a control. f, g Substrate candidates for Parkin (f) and TRIM28 (g). Relative label-free quantification (LFQ) abundance is indicated by the color scale. Proteins with PSMs of >3 in at least one experiment (Exp) and >1 in at least two experiments were considered as substrate candidates for each E3 ligase. h, i Detection of ubiquitinated endogenous substrates. Cells expressing only FLAG–TUBE, FLAG–TUBE and each E3 ligase independently or the FLAG–TUBE-fused probe were harvested and anti-FLAG immunoprecipitates were analyzed by immunoblotting. Vertical bars and arrows denote the positions of ubiquitinated substrates and unmodified substrates, respectively.