Mapping Pirh2 domain for in vitro self-ubiquitination.

<p>(<b>a</b>) Schematic representation of Pirh2 mutant constructs for the N-terminal domain, Ring domain, and C-terminal domain. (<b>b</b>) GST purified proteins run on a 10% SDS gel and stained with Coomassie blue. (<b>c</b>) In vitro ubiquitination assays of Pirh2 and Pirh2 mutant constructs. Reactions were performed in the presence of E1, E2 (H5B), and Myc-Ub. Following ubiquitination reactions (90 min), reactions were stopped by the addition of SDS loading dye and resolved on a 10% SDS-PAGE gel. Data were collected after immunoblotting with anti-Myc antibody to reveal ubiquitinated proteins and with anti-His antibody to reveal E1 and E2 proteins. (<b>d</b>) The ubiquitin integrated density of the original films was evaluated using Photoshop density quantification to analyze the ubiquitination intensity of each band.