Fig. 1

Identification of TRIM25 as an E3 ubiquitin ligase of DDX20. A IB analysis of total ubiquitination of FLAG-tagged DDX20 in 293T cells co-transfected with plasmids expressing DDX20-FLAG; immunoprecipitates were pull down by nickel beads, and the ubiquitylated target protein was detected using an anti-FLAG antibody. B IB analysis of total ubiquitination of endogenous DDX20 in 293T cells incubated with the proteasome inhibitor MG132 or not. Whole cell lysates were immunoprecipitated with anti-DDX20 antibody and blotted with an anti-Ubiquitin antibody. The working concentration of MG132 was 20 μM. C Proteins interacting with DDX20 were identified by co-IP and mass spectrometry analyses using 293T cells as a cell model. The yellow star in Group: DDX means that DDX20-FLAG was overexpressed. D MYC-TRIM25 and DDX20-FLAG were transfected into 293T cells for 48 h, and then immunoprecipitation assays were performed with an anti-FLAG antibody, whereas MYC-TRIM25, DDX20-FLAG and GAPDH were detected with the indicated antibodies respectively. E Upper: Schematic representation of GFP tag fused (GFP-TRIM25) and mCherry tag fused DDX20(mCherry-DDX20); Lower: 293T cells were co-transfected with GFP-TRIM25 and DDX20-mCherry, the co-localization of GFP-TRIM25(green) and DDX20-mCherry (red) were detected. Scale bar denotes 10 μm. F 293T cells expressing HIS-Ubiquitin and DDX20-FLAG were transfected with MYC-TRIM25 or the empty myc-vector for 48 h. HIS ubiquitylation assays were performed as described in Materials and Methods. Left: The ubiquitylation level of DDX20-FLAG was detected using an anti-FLAG antibody. Right: Quantitative analysis of relative ubiquitinated DDX20-FLAG in cell expressing MYC-TRIM25 or not. G 293T cells were co-transfected with DDX20-FLAG and HIS-Ubiquitin together with anti-TRIM25 shRNA for 48 h. the polyubiquitination level of DDX20-FLAG was detected. INPUT: Whole cell lysate, IP: immunoprecipitation, PD: pull down, IB: immunoblot. Relative ubiquitination level of target protein = gray value of ubiquitination band in each lane/(gray value of target protein/gray value of internal reference gene). *p < 0.05, between indicated groups. Data are shown as the mean ± SD of three independent experiments