A216T and L217F mutants of PML show deficiencies to SUMO and ubiquitin conjugation in response to arsenic. (A) Overview of the experimental design for detection of ubiquitin and SUMO conjugated to PML. (B) Schematic depiction of the expected contents of YFP-PML-V bound to beads and resulting supernatants after treatment with specific proteases. (C) Anti-PML immunoblot for nuclear extracts from the indicated cell lines before and after purification of YFP-PML-V with anti-GFP nanobody beads. (D and E) Analysis of the material remaining bound to anti-GFP nanobody beads (D) or eluted from the beads (E) by treatments of purified PML bodies with SUMO and ubiquitin-specific proteases. Immunoblotting used antibodies to PML, SUMO1, SUMO2/3, or ubiquitin. 2 ng standards of recombinant proteins (Rec.) were included in the eluted proteins analysis (E). The positions of recombinant PML (“R-PML”) and unmodified forms of YFP-PML (“0” or “Y-PML”) are indicated. Higher molecular weight modified forms of YFP-PML-V are indicated (“+1,” “+2,” “+3”), and ubiquitin-modified SUMO molecules are indicated with asterisks (“*,” “**,” “***”). Source data are available for this figure: SourceData F2.