Fig. 2: T194/G195 Split-TurboID is suitable for SUMO-ID studies.

a Structure of the E. coli BirA with bound biotin (PDB ID: 1HXD^80,82), depicting the T194/G195 split point and the BirA-Biotin interaction. T194/G195 split point breaks the 193-199 loop that connects the biotin-interacting β8 and β9 strands (see Supplementary Note 1). b Western blot of HEK293FT cells that were transiently transfected with combinations of the FLAG-N or MYC-C fused to PML, RANGAP1 or SUMO1/2 and treated with 50 μM of biotin for 16 h. Black arrowheads indicate SUMO-ID activity derived from MYC-C-SUMOylated FLAG-N-substrates. White squares indicate biotinylated free MYC-C-SUMOs. Neither FLAG-N nor MYC-C showed any detectable background biotinylating activity. Data are representative of two independent transfection experiments with similar results. Source data are provided in the Source Data file. c Immunostainings of transiently transfected U2OS cells treated with 50 μM of biotin for 16 h, showing the fragment-complementation dependency of SUMO-ID and its correct localization within the cell, enriched at PML NBs as expected for SUMOylated PML. Nuclei are stained with DAPI (blue) and biotinylated material with fluorescent streptavidin (Strep, magenta). BirA antibody recognizes both N and C (green). Black and white panels show the single green and magenta channels. Scale bar: 5 μm. Images are representative of two independent transfection experiments performed on cover slips. d Western blot of HEK293FT stable cells for N-FRB in combination with C-FKBP, treated or not with 1 μg/mL of rapamycin and 50 μM of biotin at indicated time-points. BirA antibody recognizes both N and C. White squares and circles indicate N-FRB and C-FKBP, respectively. Self-biotinylating activity of the reconstituted TurboID was measured and normalized to expression levels (BirA blot). Bar plots show the mean and standard deviation of three independent experiments. Statistical analyses were performed by 2-way ANOVA: *p < 0.05; **p < 0.01; ****p < 0.0001. Molecular weight markers are shown to the left of the blots in kDa. Source data and the exact p values are provided in the Source Data file.