(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).