Fig. 2: Tandem connection of UBA domains increases the efficiency of identification of substrate candidates. | Communications Biology

Fig. 2: Tandem connection of UBA domains increases the efficiency of identification of substrate candidates.

From: A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets

Fig. 2

a Construction of an N-terminal FLAG–UBA-fused probe. b, c Venn diagrams for identified substrate candidates in Parkin experiments with CCCP (10 μM) treatment for 1 h (b) and TRIM28 experiments (c) in HEK293T cells stably expressing each probe. Probes in which TUBE or UBA was fused to the N terminus of each E3 ligase were used. d, e Substrate candidates for Parkin (d) and TRIM28 (e). 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. f, g Detection of ubiquitinated endogenous substrates. Cells expressing the FLAG–TUBE or FLAG–UBA-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.

Back to article page