Fig. 3: SUMO-ID is specific for SUMO-dependent interactions.

a Western blot of HEK293FT FLAG-N-PML stable cell line transfected with different combinations of MYC-C and SUMO^WT or SUMO^ΔGG. Cells were treated or not with 1 μM of ATO for 2 h and 50 μM of biotin at indicated time points. White square indicates biotinylation of unconjugated MYC-C- SUMO^ΔGG. White arrowhead points to SUMO-SIM interaction mediated SUMO-ID. Black arrowhead shows PML-SUMOylation derived SUMO-ID. b Western blot of HEK293FT transfected with constitutive MYC-C-SUMO1/2 or doxycycline-inducible and isopeptidase-cleavage resistant (nc) MYC-C-SUMO1/2nc or MYC-C-Ubnc. Doxycycline was added or not at 1 μg/mL for 24 h. White squares point to free/unconjugated MYC-C-SUMOs. Dotted line indicates a cut in the same blot. c Western blot of U2OS double stable cell lines for FLAG-N-PML^WT or the SUMO/SIM mutant FLAG-N-PML^3MAS together with doxycycline-inducible TRIPZ-MYC-C-SUMO2nc. Doxycycline was added or not at 1 μg/mL for 24 h. 50 μM of biotin was added at indicated time-points. PML SUMO-ID showed a high PML/SUMO interaction dependency. d Confocal microscopy of the same cells as in c, treated or not with doxycycline (1 μg/mL, 24 h), biotin (50 μM, 2 h) and ATO (1 μM, 2 h). Nuclei are stained with DAPI (blue) and biotinylated material with fluorescent streptavidin (Strep, magenta). BirA antibody shows N-PML staining (green). Black and white panels show the single green and magenta channels. Colocalization of the streptavidin and N-PML^WT signal is observed within PML NBs, that depends on PML-SUMO interaction. Scale bar: 5 μm. Images are representative of three independent experiments. a–c are representative of 2 biological replicates with similar results. Molecular weight markers are shown to the left of the blots in kDa. Source data are provided in the Source Data file.