P97 inhibition leads to the accumulation of arsenic-induced degradation intermediates. PML bodies were isolated from U2OS PML^−/− + YFP-PML cells that were either untreated (0), treated with 1 μM arsenic (As), 5 μM CB-5083 (CB), or a combination of arsenic and CB-5083 (As/CB). Purified PML bodies bound to anti-GFP nanobody magnetic beads were either untreated (no proteases), treated with SUMO specific protease SENP1 (+SENP1), treated with ubiquitin-specific protease USP2 (+USP2), or a combination of both (+USP2+SENP1). (A–F) Bound material was eluted and analyzed by Western blotting using antibodies to PML (A), SUMO1 (B), SUMO2 (C), ubiquitin (D), K48-linked ubiquitin (E), K63-linked ubiquitin (F). Molecular weight markers are indicated in kD. (G) Relative intensity in the indicated proteomics samples of peptides characteristic of Ub-Ub branch points at K48 (left) and K63 (right). Bars show average intensity and errors are 1 SD. P values from Students two-tailed t test (*with Welch’s correction) are shown above bars. n = 4 independent experiments (see Fig. 1 A). Source data are available for this figure: SourceData F6.