Pirh2 mediated p53 ubiquitination mainly through the K48 chain of ubiquitin in vitro.

<p>(<b>a</b>) In vivo self-ubiquitination reactions were performed in H1299 cells. Cells were transfected with plasmids expressing WT Pirh2 or the mutant constructs illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082803#pone-0082803-g002" target="_blank">Figure 2</a> and analyzed by Western blotting using anti-HA antibodies to reveal ubiquitinated proteins (top) and using anti-GST antibodies to detect Pirh2 WT or mutant proteins (bottom). Actin expression was used as a loading control. <b>(b) and (c) Pirh2-p53 </b><b><i>in-vitro</i></b><b> ubiquitination using human (b) and mouse (c) derived proteins.</b> Ubiquitination reactions were performed in the presence of GST-Pirh2, His purified p53, E1, E2 (H5B), and Ub-Wt. Pirh2 human and mouse derived proteins were used in three different concentrations (300, 600, and 900 ng). Following ubiquitination reactions (90 min), reactions were stopped by the addition of SDS loading dye and resolved on a 10% SDS-PAGE gel, and then, immunoblotting was performed using anti-p53 antibodies to reveal ubiquitinated p53 proteins. DO-1 was the most efficient in detecting human p53 ubiquitination (<b>b</b>). P122 was highly efficient in detecting mouse p53 ubiquitination (<b>c</b>). Anti-GST antibodies were used for Pirh2 detection as indicated. (<b>d</b>) Pirh2 ubiquitination using WT-Ub, Ub-K63R, Ub-K48R, or Ub-KO in the presence or absence of p53. 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-ubiquitin antibodies to reveal ubiquitinated proteins, with DO-1 anti-p53 antibody to reveal p53, with anti-His antibody to reveal E1 and E2, and with anti-GST antibody to detect Pirh2. (<b>e</b>) The ubiquitin integrated density of the original films was evaluated using Photoshop density quantification to analyze the ubiquitination intensity of each reaction. (<b>f</b>) p53 in vitro ubiquitination by Pirh2 using the four ubiquitin constructs described above. Immunoblotting was performed using anti-p53 antibody (Do-1) to reveal ubiquitinated p53 protein. Anti-His antibody was used to detect E1 and E2 and anti-GST antibody to detect Pirh2.