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. Author manuscript; available in PMC: 2010 Oct 9.
Published in final edited form as: Mol Cell. 2009 Oct 9;36(1):28–38. doi: 10.1016/j.molcel.2009.09.016

Figure 2. The otubain domain confers catalytic activity independent of accessory domains.

Figure 2

(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).