Fig. 8

Tripartite motif containing 32 (TRIM32) is a Z-DNA binding protein 1 (ZBP1) E3 ubiquitin (Ub) ligase destabilizing ZBP1. a Western blotting analysis showing the expression of Flag-tagged ZBP1 in endothelial cells transfected with a Flag-ZBP1 plasmid, indicating a significant overexpression of ZBP1 compared to the control group. Quantification of the ZBP1 expression level is presented in the bar graph below with a significant increase in ZBP1 expression. b Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of ZBP1-interacting proteins identified through ZBP1 immunoprecipitation followed by mass spectrometry from endothelial cells expressing ZBP1. The graph displays the enriched pathways, with Ub-mediated proteolysis being the most significant. c Co-IP assay from endothelial cell lysates using an anti-ZBP1 antibody, followed by immunoblotting for TRIM32 and ZBP1. The input and IP samples demonstrate the interaction between ZBP1 and TRIM32. d Lung vascular endothelial cells were transfected with a TRIM32 plasmid, and cell lysates were immunoprecipitated with an anti-TRIM32 antibody, followed by ZBP1 immunoblotting. e A GST-pulldown assay confirming a direct interaction between GST-tagged TRIM32 and ZBP1, as indicated by the presence of ZBP1 in the pulldown complex. f Immunofluorescence staining of TRIM32 (red) and ZBP1 (green) in endothelial cells, with nuclei counterstained with DAPI (blue). The merged image and the enlarged panel from the highlighted box show the colocalization of TRIM32 and ZBP1 (scale bar = 20 μm). Line graphs on the right display fluorescence intensity profiles for ZBP1 and TRIM32, corroborating their colocalization within the cells. The arrow from “i” to “ii” highlights the region analyzed for colocalization analysis. g Stability of ZBP1 protein was assessed over time by Western blotting in control cells, cells expressing GST-TRIM32 and TRIM32 knockdown, treated with cycloheximide (CHX) to inhibit new protein synthesis. h The plot below the blots quantifies ZBP1 levels, revealing the effects of GST-TRIM32 overexpression and siTRIM32 knockdown on ZBP1 stability. i Western blotting analysis of ZBP1 levels in the presence of CHX with and without MG132 treatment in the GST-TRIM32 overexpression group. The symbols *, **, and *** indicate statistically significant differences compared to the control group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. j Ubiquitination assay depicting the modification of ZBP1 in the presence of HA-tagged Ub and GST-TRIM32. The smear of high molecular weight bands above ZBP1 indicates polyubiquitination. The assay was conducted using an anti-Flag antibody for immunoprecipitation, and ubiquitination levels were assessed by anti-HA Western blotting. Expression levels of TRIM32 and ZBP1 were verified by analyzing 5% of the input from cell lysates. The data are presented as the mean ± SD. MG132 a proteasome inhibitor, Co-IP co-immunoprecipitation