Noc 5

We assessed the concentration-dependent effects of nitric oxide on UBE2D3 activity employing an in vitro ubiquitination assay (MDM2 Ubiquitin Ligase Assay - p53 Substrate, BostonBiochem), in which UBE2D3 activates the Ring finger E3 ubiquitin ligase, MDM2 (murine double minute 2), which in turn conjugates ubiquitin with its substrate, His⁶-p53. Ubiquitination of the latter is subsequently quantified per Western Blot analysis with anti-His or anti-p53 antibody detection. The assay employs recombinant proteins for E1, UBE2D3, GST-MDM2 and the substrate, His⁶-p53. The reaction was prepared in 30 µl on ice by adding the components one by one in the absence and presence of 0.1–100 µM NO donors, DEA/NO and NOC-5 (both Sigma, added after UBE2D3), and finally started by adding ubiquitin. After 1 h incubation, the reaction was terminated by adding 8 µl 5X SDS-PAGE sample buffer and 2 µl 1 M DTT. Samples were directly submitted to standard 10% SDS-PAGE, electro-blotting onto nitrocellulose membranes and detection with anti-p53/anti mouse-IR-dye on an Odyssee infrared scanner.