Fig. 3: USP18 promotes K27-linked ubiquitination of TRIM29 independent of its catalytic activity.

A Denatured-IP assay (anti-Myc) and western blotting (anti-Myc, HA, and α-Tubulin) in HEK293T and SUNE1 cells transfected with TRIM29-Myc, HA-Ub, and either vector, USP18-Flag WT, or C64S mutant. B Denatured-IP assay (anti-Myc) and western blot analysis (anti-Myc, HA, USP18, and α-Tubulin) in USP18-WT and USP18-KO SUNE1 cells transfected with TRIM29-Myc and HA-Ub. C Denatured-IP assay (anti-Myc) and western blot analysis (anti-Myc, HA, and α-Tubulin) in HEK293T cells transfected with TRIM29-Myc along with vector or USP18-Flag plus HA-Ub-WT or HA-Ub-KO mutants retaining only specific tyrosine sites of ubiquitin (K6O, K11O, K27O, K29O, K33O, K48O, and H63O). D K27O-linked ubiquitination level of TRIM29-Myc in USP18-KO SUNE1 cells supplemented with vector, USP18-Flag WT, or C64S mutant. E Ubiquitination levels of TRIM29-Myc truncations in HEK293T cells co-transfected with USP18-Flag and HA-Ub. F Mass spectrometry analysis of TRIM29 ubiquitination sites in SUNE1 cells co-transfected with TRIM29-Myc and HA-Ub-K27O. G Ubiquitination levels of TRIM29-Myc WT and KR mutants in HEK293T cells transfected with USP18-Flag and HA-Ub-K27O. H Mass spectrum of the K561 site on TRIM29. I Sequence alignment of the K561 site within TRIM29 orthologs across species. Results are representative of three independent experiments A–E, G. The unprocessed images of the blots are shown in Fig. S11.