Additional figures of MBP-OtUBD and immobilized OtUBD-resin pulldowns.

(A, B) Ubiquitin pulldowns with different amounts of MBP-OtUBD. In A, the pulldown was performed by first binding MBP-OtUBD to an amylose resin and then incubating the resin with yeast cell lysate. In B, pulldown was performed by incubating the lysate with MBP-OtUBD and then binding the complexes to amylose resin. U, unbound fraction; E, fraction eluted with maltose. The bands seen at the expected molecular mass of MBP-UBD were likely a result of antibody cross-reactivity. (C) Anti-ubiquitin blot of MBP-OtUBD pulldowns from HEK293T whole cell lysates. U, unbound fraction; B, bound fraction (eluted with SDS sample buffer). (D) SYPRO Ruby protein stain of the eluates from OtUBD-resin in Fig 2B. E1/E2/E3, eluted fractions from serial low pH elutions. MBP, maltose-binding protein.

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