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. 2007 Aug 6;27(19):6962–6971. doi: 10.1128/MCB.02415-06

FIG. 7.

FIG. 7.

Polybiquitination of Bach1 in vitro. (A) Recombinant Bach1 (wild type [WT]) or Bach1mCP1-6 containing mutations in the six CP motifs (M) were incubated with ubiquitin aldehyde, ubiquitin, ATP and the ATP-regenerating system in the presence (+) or absence (−) of combinations of E1, E2 (UbcH5c), and HOIL-1 as indicated. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel. IB : Bach1, immunoblotting with anti-Bach1 antibody; (Ub)n-Bach1, polyubiquitinated Bach1; NS, nonspecific. (B) Ubiquitination reactions of wild-type and Bach1mCP1-6 (0.5 μg) were carried out as described above for panel A in the presence of the indicated concentrations of hemin. HOIL-1 together with E1 and E2 enzymes efficiently monoubiquitinated Bach1 (indicated by the arrow). The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel.