a, Ub-AMC hydrolysis assay of Usp14 activity in the presence or absence of Ub-VS treated human proteasome (VS-proteasome; 1 nM). RFU, relative fluorescence units. Ptsm, 26S proteasome.
b, In vitro degradation assay with polyubiquitinated cyclin B (Ub_n-ClnB), human proteasome (4 nM), and wild-type Usp14 (Usp14-wt) or mutant Usp14-CA (60 nM). Samples in b, c, and e-h analyzed by SDS-PAGE/immunoblotting (IB).
c, Plasmids expressing Tau, TDP-43^Flag, or LacZ^V5 were cotransfected into usp14^−/− MEFs with variants of Usp14^Flag as indicated. Samples collected 2 days post-transfection. Actin, load control.
d, Diagram of human Usp14, showing ubiquitin-like (UBL) and catalytic (CAT) domains. C114, active site cysteine. Splice variant Usp14-SF is produced from an mRNA lacking exon 4 (ref 12).
e, Flag-tagged Ataxin3-Q22 or -Q80 was coexpressed with Usp14 variants in usp14^−/− MEFs and detected with anti-Flag antibodies.
f, Arg-GFP or control Met-GFP coexpressed with Usp14 variants in usp14^−/− MEFs.
g, As c except in HEK293 cells.
h, Usp14-SF associates with but is not activated by proteasomes. Each variant of Usp14^Flag was expressed in HEK293T cells containing tagged hRpn11, and proteasomes were affinity purified. Where indicated, Ub-VS was incubated with lysate prior to proteasome purification. Extract samples represent 5% of total. Asterisks, nonspecific signals. Proteasome subunit Rpn13, load control. Control samples, empty vector. Equal cell numbers were used for each lane.

a, Chemical structures of IU1 and IU1C. Analytical data shown in Supplementary Fig. 16.
b, IC₅₀ determination for IU1 inhibition of Ub-AMC hydrolysis by proteasome-bound Usp14 (4.7 ± 0.7 μM), IsoT (100 ± 0.4μM), and Uch37 (0.7 ± 0.3 mM).
c, Ub-AMC (1 μM) hydrolysis assays showing specificity of IU1 for Usp14.
d, Reversibility of Usp14 inhibition. 60 nM Usp14 and 5 nM human proteasome were treated with vehicle (DMSO) or 100 μM IU1 for 2 hr. After spin gel-filtration, proteins were assayed for Ub-AMC hydrolysis. All values are presented as mean ± s.d. (n=3).

a, Chain trimming assays. Samples contained 4 nM proteasome, and Usp14 was added at 15-fold molar excess over proteasome. IU1 was added at 50 μM and proteasome inhibitors (PI) at 5 μM (PS-341, epoxomicin). Asterisk, cyclin B species derived from residual thrombin from Usp14 preparation16. All panels, SDS-PAGE/immunoblot analysis.
b, In vitro Ub_n-ClnB degradation assay (IU1 at 34 μM).
c, In vitro degradation assay with polyubiquitinated Sic1^PY, human proteasome (5 nM), and Usp14-wt (75 nM) in the absence or presence of IU1 (75 μM).

All panels show SDS-PAGE/immunoblot data.
a, 36 hours after cotransfecting wild-type MEFs with plasmids expressing Tau and LacZ^V5, cells were incubated with 0, 25, 50, 75, or 100 μM of IU1 for 6 hr. LacZ, transfection control. Actin, loading control.
b, As in a except that MEFs were usp14^−/− and IU1 was at 0, 10, 50, or 100 μM.
c, Tau and Ub-independent proteasome substrate cODC-EGFP were coexpressed in wild-type MEFs and incubated with 50 μM IU1 for 6hr. Proteasome inhibitors were MG132 (30 μM) and PS-341 (10μM).
d, As b except with Atx3-Q80 and Atx3-Q22.
e, TDP-43^Flag was cotransfected with a LacZ-expressing plasmid into either wild-type or usp14^−/− MEFs, then treated with IU1 (75 μM) for the time indicated. Asterisk, nonspecific signal.
f, HA-tagged Ub and/or Flag-tagged TDP-43 were transiently overexpressed in wild-type MEFs with 50μM IU1 incubation for 6 hr. Proteasome inhibitors (20 μM MG132, 10 μM PS-341) were added 4 hr before lysis. Lysates were subjected to immunoprecipitation with anti-HA or anti-Flag. Arrows indicate likely ubiquitinated TDP-43 species. HC, heavy chain.
g, Wild-type MEF and usp14^−/− MEF cells were treated with IU1 (0, 25, 50, 75, or 100 μM) for 6 hr, followed by analysis for ubiquitin, actin, CP subunit α7, and RP subunit Rpt5.

a, HEK293 cells were preincubated with IU1 (75 μM) or proteasome inhibitors (20 μM MG132, 10 μM PS-341) for 4 hr, then treated with menadione (300 μM) for 60 min. Lysates were treated with DNPH and immunoblotted with anti-DNP antibody to assay oxidized proteins.
b, Cell survival under oxidative stress measured using the MTT assay. HEK293 cells were pretreated with 50 μM IU1 for 2 hr. Menadione was added, followed by 4-hr incubation. IU1 effects comparable to those of panels a and b were obtained in wild-type but not usp14^−/− MEFs (data not shown). Values are represented as mean ± s.d. (n=3).