Figure 5. Effect of Siah2 inhibitory peptides on Siah2 ubiquitination and activity in vitro and in cell.
a) In vitro self-ubiquitination of Siah2 was performed in the presence of DMSO (control) or indicated concentrations of BI-107F7 (indicated as F7), with UB-HA and ubiquitination reaction buffer. Degree of ubiquitination was monitored using antibodies to HA. b) In vitro ubiquitination of OGDH was monitored in the presence of indicated concentrations of BI-107F7 or BI-107F9 (indicated as F7 and F9, respectively). Poly-ubiquitinated OGDH is marked on the right panel. c) BI-107F9 and BI-107F8 peptides were added to SW1 melanoma cells grown in culture, and the effect of Siah2 on exogenously expressed PHD3 was monitored. α-tubulin was monitored as a loading control. d) BI-107F9 and BI-107F10, at the indicated concentrations, were added to cultured melanoma cells maintained in 1% O2. 6h later cells were harvested and level of HIF-1α and α-tubulin were assessed.