Fig 2: DDI2 Cleaves Ubiquitylated NRF1 Protein In Vitro(A) Western blot analysis of NRF1 protein, isolated via GST-DSK2 chromatography, after treatment with DDI2, RAD23, and USP2, as indicated (4%–15% TGX). The mobility of NRF1 and cleaved NRF1 is shown on the right (see also arrow for cleaved NRF1). Zoomed-in images of lanes 13 and 14 and 19 and 20 are shown below. Note that large amounts of extract were used as input for GST-DSK2 purification, meaning that in relative terms, ~20× “extract equivalents” were loaded in lanes 5–20 compared to lanes 1–4. See also Figure S6A.(B) Reprobing of the membrane from (A), with anti-ubiquitin antibodies. For simplicity, only the relevant lanes 5–9 are shown.(C) Experiment as in (A), but testing DDI2D?N, as indicated.(D) As in (A) and (B), but testing cleavage of exogenously expressed, Myc-tagged versions of NRF1. N, normal NRF1 sequence. m, mutated sequence. Lower panel, lanes 2 and 6 are shown in the enlargement. The “dot” in lane 6 is not a cleavage band.See also independent experiment in Figure S6B.

Fig 3: DDI2 Fails to Cleave Purified Ubiquitin Chains but Preferentially Binds and Cleaves Slowly Migrating Ubiquitylated Species(A) Coomassie-stained gels of different commercially available ubiquitin chains before and after incubation with DDI2, RAD23, or the catalytic domain of USP2, as indicated. Untreated substrates are indicated by stippled boxes. Note that USP2 cleaves all these chains to mono- or di-ubiquitin. See also lack of detectable mono-/di-ubiquitin in the blot of Figure S5D.(B) Schematic of experiments in (C) and (D).(C) Western blot analysis of ubiquitylated proteins in bound and unbound (supernatant) fractions after incubation with chemically inactivated DDI2 protein (note that two biological replicates of the same experiment are shown; lanes 1 and 2/5 and 6 and lanes 3 and 4/7 and 8). Note the specific depletion of slowly migrating ubiquitylated proteins in lanes 1 and 3 and the enrichment of the same in lanes 5 and 7.(D) As in (C), but ubiquitylated proteins on FLAG-DDI2D?N beads, incubated with DDI2 or DDI2D?N (non-FLAG tagged to avoid non-specific “displacement” from the beads), as indicated. Note the disappearance of ubiquitylated proteins from beads after incubation with WT DDI2 (above black stippled line, lane 3) and concomitant release of faster-migrating ubiquitylated proteins into the supernatant (below red stippled line, lane 9). See also ImageJ scanning traces below, with position of stippled lines indicated for reference. Please note that the exposure time of the blot on the right is longer than that on the left; only small amounts DDI2-bound material can be eluted.See also Figure S5.

Fig 4: Increased RIPK3 ubiquitination in USP22 KO HT-29 cells during TBZ-induced necroptosis HT-29 control, USP22 KO, and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 WT or C185S were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFa for 4 h. Poly-ubiquitinated proteins were enriched by GST-TUBE pull-down, followed by incubation with the catalytic domain of USP2, as indicated. RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies. ß-Actin served as loading control. Ponceau staining was used to confirm equal loading of GST-TUBE.HT-29 control and USP22 KO cells were transfected with His-ubiquitin for 24 h, as indicated. Cells were pre-stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 for 1 h. Following pre-treatment, 1 ng/ml TNFa was added for 4 h. His-ubiquitin was immunoprecipitated using Ni-NTA beads, and detection of indicated proteins was performed by Western blotting. ß-Actin served as loading control for the input, whereas His-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-stimulated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. Following pre-treatment, 10 ng/ml TNFa were added for 3 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads, and detection of indicated proteins was performed by Western blotting. ß-Actin served as loading control for the input, whereas HA-levels served as loading control for immunoprecipitated ubiquitin.

Fig 5: Anti-HA Western Blot analysis of the labelling of selected recombinant DUBs (OTUB2, USP2 and USP7) with HA-1-75Ub-alkene probe 1.HA-1-75Ub-alkene probe 1 (2 μg) was incubated with recombinant DUBs OTUB2 (1 μg), USP2 (2 μg) and USP7 (5 μg), for 90 min at 37 °C, prior to the addition of Irgacure 2959 (250 μM, 2-min UV irradiation), Mes-Acr+ (100 μM, 10-min blue light irradiation) or Mn(OAc)3 (125 μM, 30-min incubation at 37 °C). A negative control without initiator was performed. Results were analysed by SDS-PAGE and visualised by anti-HA Western Blot.