Figure 12
In vitro ubiquitination. The reactions, lacking a defined reactant, were incubated at 37°C for 3 h, and assayed for ubiquitin ligase activities. The reactions were terminated by adding an equal volume of SDS-loading dye before electrophoresis on 10% SDS/PAGE. The separated protein was then transferred to a PVDF membrane and immunodetected with conjugated anti-His 6 Horseradish Peroxidase. The blot was detected by chemiluminescence on an X-ray film (see materials and methods). The loading control of E3 ligase activity and an apparent ubiquitination product was indicated by filled diamond (A). The effects of 1 and 2 on E3 ligase activity were compared with the non-treated control BRCA1 (B). The relative E3 ligase activity of the BRCA1 adducts was quantified by normalizing the density of an apparent band of the ubiquitinated-protein conjugates to that of the parental BRCA1 as the control, using a Bio-Rad GS-700 Imaging Densitometer. The relative E3 ligase activity of the BRCA1 adducts (%) was plotted as a function of the concentration of the ruthenium(II) polypyridyl complexes (C). The significant reduction of E3 ligase activity of the BRCA1 adducts induced by 1 or 2 was compared using one-way ANOVA (* = p < 0.01), relative to control.