Fig. 8: The in vitro DUB activity of OTU11 and OTU12 is stimulated by anionic lipids.

a, b DUB assays with GST-OTU11 (a) and GST-OTU12 (b). 7.5 pmol of K63-linked tetra-UB was incubated with 7.5 pmol (substrate: enzyme ratio 1:1), 25 pmol (1:3), 50 pmol (1:6), 100 pmol (1:12), or 250 pmol (1:30) of GST-OTU11 or GST-OTU12 for 1 h at 21 °C. The experiments were repeated at least three times, and one representative image is shown. c DUB assay with phosphatase treated GFP-OTU11 purified from 35Spro:GFP-OTU11 expressing Arabidopsis seedlings and 15 pmol of K63-linked di-UB. active: active phosphatase, inact: heat-inactivated phosphatase. The experiment was repeated at least three times; one representative image is shown. d, e DUB assays with GST-OTU11 (WT) and GST-OTU11(6A1) pre-incubated with liposomes generated with PC and PE (PC/PE) alone (−PIP) or with liposomes (PC/PE) containing 5% PI(4,5)P₂ (+PIP). In (d), 25 pmol of GST-OTU11(WT) or GST-OTU11(6A1) was incubated for the indicated time with 25 pmol of K63-linked tetra-UB (negative controls: tetra-UB with +PIP or −PIP liposomes, incubated for 4 h). In (e), 25 pmol of GST-OTU11, pre-incubated with +PIP and -PIP liposomes, was mixed with 25 pmol of linear, K6-, K11-, K27-, K29-, K33-, or K48-linked tetra-UB for 4 h at 21 °C. The experiments were repeated at least three times; one representative image is shown. f–k Effect of liposomes on the DUB activity of OTU11 (f–h) and OTU12 (i–k). 3.75 pmol of Recombinant OTU11(WT), OTU11(6A1), OTU12(WT), and OTU12(6A1) were pre-incubated with either liposome with or without PI(4,5)P₂ [lipo(+PIP) and lipo(–PIP), respectively], or the liposome buffer alone before the addition of 3.75 pmol of di-UB FRET TAMRA K48 pos1 (f, i), K48 pos2 (g, j), and K63 pos1 (h, k). The assays were conducted at least three times. The result of one representative measurement is shown. Error bars: standard deviation of a technical quadruplicate, center of the error bars: mean of the quadruplicates. Source data are provided as a Source Data file.