(A) Heterologously expressed, purified YOD1 variants (10 μg each) were separated by SDS-PAGE (12%) and stained with Coomassie Blue.
(B) K48-linked poly-Ub chains (2 μg) were incubated for 16 h in a total volume of 10 μl with different YOD1 variants (7.7 μM). Poly-Ub and free Ub were detected by immunoblotting using anti-Ub antibodies.
(C) K48-linked di-Ub (0.5 μg) were incubated for indicated times at 25°C in a total volume of 10 μl with different YOD1 variants (2.58 μM). Ub was detected by immunoblotting using anti-Ub antibodies.
(D) K48-, K63-linked and genetically fused linear di-Ub (2 μg) were incubated for 16 h in a total volume of 10 μl with various YOD1 variants (5.2 μM), and detected as in (B).

(A) 293T cells were transfected with RI₃₃₂ and either full-length or truncated (ΔUBX) YOD1 WT or C160S. The experiment was performed as in Fig. 3 A.
(B) Quantification of (A). The error bars represent the standard deviation of three independent experiments. The asterisk indicates a proteolytic fragment of ΔUBX YOD1.
(C) 293T cells were transfected with either full-length or truncated (ΔZnf) YOD1 WT or C160S. The experiment was performed as in Fig. 3 A.
(D) Quantification of (C). The error bars represent the deviation from the mean of two independent experiments.

(A) 293T cells transiently transfected with RI₃₃₂, HA-ubiquitin and either YOD1 WT or C160S were lysed in NP40 lysis buffer, and immunoprecipitated with anti-RI antibodies. The eluates were visualized by immunoblotting with anti-HA antibodies.
(B) The inpute lysates from A were directly immunoblotted and probed with anti-HA antibodies for ubiquitin levels, anti-FLAG antibodies for YOD1 levels and anti-PDI antibodies as loading control. See Fig. S5A for additional loading controls.
(C) 293T cell were transiently transfected with HA-ubiquitin, p97 WT or p97 QQ, and either empty vector (pcDNA), YOD1 WT or YOD1 C160S as indicated. After NP40 lysis and immunoprecipitation with anti-p97 antibodies the eluates were immunoblotted and probed with anti-HA antibodies. The corresponding input lysates are shown in the supplementary materials (Fig. S5 B).

(A) Domain organization of YOD1 and its mutant derivatives.
(B) 293T cells were transiently transfected with the indicated constructs or empty vector and homogenized by NP40 detergent lysis 24h post transfection. Lysates were subjected to immunoblotting with anti-FLAG and anti-p97 antibodies to control for expression of YOD1 derivatives and p97, respectively (upper and middle panels). Retrieved p97 in anti-FLAG immunoprecipitates was detected by immunoblotting with anti-p97 antibodies (lower panel).

(A) 293T cells were co-transfected with RI₃₃₂ and empty vector, YOD1 WT or YOD1 C160S. 24 hours after transfection, cells were pulse-labeled with ³⁵S for 10 min, chased for the indicated time points, lysed in 1% SDS, and the lysates were subjected to immunoprecipitation with anti-ribophorin antibodies. The eluates were resolved by 12% SDS-PAGE and visualized by autoradiography (upper panel). Unbound material was immunoprecipitated with anti-FLAG antibodies to verify equal expression of the YOD1 constructs (lower panel).
(B) Densiometric quantification of RI₃₃₂ levels. Plotted are the mean values from three independent experiments. Error bars depict the standard deviation.
(C) 293T cells were co-transfected with a cytosolic variant of RI₃₃₂ lacking its N-terminal signal sequence (ΔSS RI₃₃₂) and with either empty vector (pcDNA), YOD1 WT or catalytically inactive YOD1 C160S, and processed as in (A).
(D) ΔSS RI₃₃₂ stability was quantified as in (B).
(E) Cells co-transfected with YOD1 C160S and RI₃₃₂ were metabolically labeled as in (A). Cell extracts were prepared in hypotonic buffer by homogenization in absence of detergent. The homogenate was incubated on ice in presence and absence of 100 μg/ml proteinase K. 0.5% NP40 was added as indicated. Proteinase K was inactivated after 15 min by inclusion of PMSF. The resulting material was solubilized with 1% SDS, immunoprecipitated with anti-ribophorin antibodies and processed as in (A).

(A) 293T were cells transfected with α1-antitrypsin NHK (α1 AT), and YOD1 WT or YOD1 C160S, were pulse-labeled with ³⁵S for 10 min and chased for the indicated time points. The cells were lysed in SDS and the lysates were immunoprecipitated with anti-α1-AT antibodies. The eluates were separated by SDS PAGE (12%) and the bands were visualized by autoradiography.
(B) Quantification of NHK levels. Plotted is the mean value of three independent experiments with the error bar corresponding to the standard deviation.
(C) 293T cells were transfected with TCRα, and either empty vector (pcDNA), YOD1 WT or YOD1 C160S. The pulse-chase experiment was performed as in Fig. 3 A. TCRα was retrieved from SDS-lysates by immunoprecipitation with anti-TCRα antibodies and visualized by autoradiography.
(D) Quantification of TCRα levels. Plotted is the mean value of two independent experiments with error bars.

(A) 293T cells were transfected with the indicated YOD1 constructs or empty vector as control and lysed in NP40. After immunoprecipitation with anti-FLAG antibodies the eluates were subjected to immunoblotting with anti-Derlin-1 antibodies.
(B) Cell lysates were prepared as in (A). Eluates were subjected to immunoblotting with anti-UBXD8 and anti-UBXD2 antibodies.