Fig. 4: USP18 recruits TRIM21 to increase K27-linked ubiquitin chains on TRIM29.
A Venn diagram showing common genes from mass spectrometry (MS) analysis of immunoprecipitated proteins from USP18-Flag and TRIM29-Myc in SUNE1 cells, along with E3 ligases from the iUUCD database. It indicates the number of TRIM21, DTX3L, and HERC5 peptides identified. B Co-immunoprecipitation (Co-IP) using anti-TRIM21 antibody in SUNE1 and HK1 cells demonstrated interactions among endogenous TRIM21, USP18, and TRIM29. C FRET assay in HEK293T cells transfected with TRIM21-YFP and TRIM29-CFP, showing representative cell images and the region used for FRET analysis. FRET efficacy (EFRET) is reported as mean ± SEM (n = 10). Scale bar: 2 μm. D Schematic representation of TRIM21-Flag truncation mutants. E Co-IP (anti-Flag) and western blot analysis (anti-Flag, HA, and α-Tubulin) in SUNE1 cells transfected with TRIM21-Flag truncation mutants and USP18-HA. F Co-IP (anti-Flag) and western blot analysis (anti-Flag, TRIM29, and GAPDH) in SUNE1 cells transfected with TRIM21-Flag truncation mutants. G Molecular docking of USP18, TRIM21, and TRIM29 as a ternary complex, with predicted amino acid linkages shown as dotted lines. H Co-IP (anti-Flag) and western blot analysis (anti-Flag, HA, USP18, and GAPDH) in WT or USP18-KO SUNE1 and HK1 cells co-transfected with TRIM21-Flag and TRIM29-HA. I Denatured-IP assay (anti-Myc) and western blotting analysis (anti-Myc, HA, Flag, and GAPDH) in HEK293T and SUNE1 cells transfected with TRIM29-Myc and HA-Ub-K27O plus vector, TRIM21-Flag WT, or C16A mutant. J, K Denatured-IP assay (anti-Myc) and western blotting analysis (anti-Myc, HA, Flag, and GAPDH) in TRIM21-KO SUNE1 cells transfected with TRIM29-Myc and HA-Ub-K27O plus vector or TRIM21-Flag WT or C16A mutant J or USP18-Flag WT or C64S mutant K. L Denatured-IP assay (anti-Myc) and western blotting analysis (anti-Myc, HA, Flag, and GAPDH) in HEK293T cells transfected with TRIM21-Flag and HA-Ub-K27O plus TRIM29-Myc WT or KR mutants. Data are representative of three independent experiments B, E and F, H to L. The unprocessed images of the blots are shown in Fig. S11.
