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. 2017 Jan 18;7:40756. doi: 10.1038/srep40756

Figure 5. Isolation and identification of bioSUMO3-conjugates in mammalian cells.

Figure 5

(A) Western blot of pulldowns from HEK 293FT cells showing that the transcription factor SALL1 fused to YFP was SUMOylated in presence (+) of bioSUMO1 (lane 4) or bioSUMO3 (lane 5; bioSUMO1-BirA or bioSUMO3-BirA, respectively). Black arrowhead indicates the modified SALL1-YFP in the elution panel (lanes 4, 5), which is shifted in comparison to the non-modified SALL1-YFP in the input panel (white arrowhead, lanes 1–3). Molecular weight markers are shown to the left. (B,C) Partial colocalization between SALL1-YFP (green) and bioSUMO1 (purple) (bioSUMO1-BirA-UBC9) in U2OS cells (B) or with endogenous SUMO2/3 (C, purple). White arrowheads indicate colocalization. Nuclei were stained with DAPI (blue). (B’,C”) Green and purple channels are shown independently in black and white. (D) SUMOylation of PML by bioSUMO3 (bioSUMO3-BirA-GP) increases after ATO treatment. bioSUMO3-modified PML (black arrowheads) can be detected by anti-HA Western blot in the input (upper panel, lanes 1–4) and the elution (lower panel, lanes 1–4; NeutrAvidin pulldown), while the level of the non-modified form of PML is reduced (input panel, lanes 1–6; white arrowhead). Note that modification of PML by endogenous SUMO is also visible in the input panel after ATO treatment (lanes 5 and 6, grey arrowheads). Control indicates cells transfected with BirA-GP. Molecular weight markers are shown to the left. (E) Up: schematic representation of the bioSUMO3 vector for mammalian cells. Left: the Western blot shows the enrichment in bioSUMO3 conjugates in the elution panel using anti-biotin antibodies (lane 3, bracket). Arrowhead indicates free bioSUMO3. Right: bioSUMO3-modified proteins identified by nLC MS/MS on Orbitrap. (F) Validation of bioSUMO3-modified proteins. Specific antibodies against endogenous proteins were used: Ub, SIRT1, RANGAP1, PML and PARP. GAPDH is shown as a control in the input panel. In the elution panels, arrowheads indicate the modified forms of the respective proteins and the bracket indicates the ubiquitinated proteins (lanes 3). Molecular weight markers are shown to the left.