Fig. 5: Ubiquitination of TRIM29 promotes oligomerization and facilitates nuclear translocation.

A Co-IP assay (anti-Myc) and western blotting analysis (anti-Flag, HA, Myc, and α-Tubulin) in SUNE1 and HK1 cells transfected with TRIM29-HA and TRIM29-Myc, with or without USP18-Flag. B Western blot analysis (anti-Flag, TRIM29, and GAPDH) of lysates from SUNE1 and HK1 cells with or without USP18 overexpression, treated with DSS (5 mM) or EGS (5 mM) for 15 min. C Native-PAGE (top) and SDS-PAGE (bottom) immunoblotting (anti-Flag, TRIM29, and α-Tubulin) analysis of TRIM29 aggregation in SUNE1 and HK1 cells transfected with vector or USP18-Flag. D Western blot analysis (anti-Flag, TRIM29, and GAPDH) of lysates from TRIM21-KO SUNE1 cells transfected with vector or USP18-Flag, treated with EGS (5 mM) for 15 min. E Western blot analysis (anti-Myc and GAPDH) of lysates from HEK293T cells transfected with TRIM29-Myc and its truncation mutants ΔBB and ΔCC, treated with EGS. F Native-PAGE and SDS-PAGE analysis (anti-Myc and GAPDH) of HEK293T cells transfected with TRIM29-Myc and its truncation mutants ΔBB and ΔCC. G Denatured-IP and western blot analysis demonstrated the ubiquitination levels of TRIM29-Myc and the ΔCC mutant in HEK293T cells expressing TRIM21-Flag. H EGS treatment of lysates from SUNE1 cells transfected with TRIM29-Myc WT or K561R mutant followed by immunoblot analysis (anti-Myc and GAPDH). I RMSD variation of the TRIM29 dimer model with or without ubiquitin during a 350 ns MD simulation stabilized after approximately 200 ns. J Stable structures of the TRIM29 dimer model obtained from MD simulations, with the N-terminal, C-terminal, Helix, and Link regions colored cyan, red, blue, and magenta, respectively. K Cytoplasmic and nuclear extraction from HEK293T cells transfected with TRIM29-WT, ΔBB, and ΔCC mutants, analyzed via immunoblot (anti-Myc, Fibrillarin, and α-Tubulin). L Chromatin and cytoplasmic extraction from WT and USP18-KO HK1 cells treated with or without IR (8 Gy) for 24 h, analyzed by immunoblot (anti-TRIM29, USP18, H3, and GAPDH). M Immunofluorescence staining showing TRIM29 distribution in SUNE1 or HK1 WT and USP18-KO cells treated with or without IR (8 Gy) for 24 h. The nuclear/cytoplasmic fluorescence ratio was calculated using ImageJ software. Scale bars: 5 μm, n = 20. N Immunoblot analysis of cytoplasmic and nuclear proteins from SUNE1 or TRIM21-KO SUNE1 cells transfected with or without USP18-Flag (anti-TRIM29, Flag, Fibrillarin, and α-Tubulin). O Immunoblot analysis of cytoplasmic and nuclear proteins from SUNE1 cells transfected with TRIM21-Flag WT. P Immunoblot analysis of cytoplasmic and nuclear proteins from SUNE1 cells transfected with TRIM29-Myc WT or K561R. Data are representative of three independent experiments A–H, K, L, N–P. Results are presented as mean ± SD; ***p < 0.001, ns: not significant; p values were determined using two-way ANOVA. The unprocessed images of the blots are shown in Fig. S11.