Figure 1.

(A) Inhibition of Siah2 auto-ubiquitination by positive hits from Meso Scale screen. In vitro ubiquitination reactions for GST-Siah2 and GST-RNF5 were carried out using 100 µM of compounds 1–4 (Table S1 and Table 1). The ubiquitination was monitored by Western blot analysis using anti-ubiquitin antibodies. The position of the ubiquitinated GST-Siah2 or GST-RNF5 is indicated. The amount of GST-Siah2 or GST-RNF5 used in these reactions is shown in the lower panel using anti-GST antibodies. (B) Increase in Siah2 steady state levels by MEN. 293T cells were transfected with HA-Siah2 (1 µg) and 24 h later cells were treated with indicated concentrations of MEN and USD 10 for 5 h. As control cells were treated with equal amounts of DMSO, Western blot analysis was performed using the indicated antibodies. β-actin level is shown as loading control. (C) Inhibition of Siah2 auto-ubiquitination by MEN. In vitro ubiquitination reactions for GST-Siah2, GST-Siah2 RING mutant (GST-S2RM) and GST-RNF5, attached to the glutathione beads, were performed using indicated concentrations of MEN. Western blot analysis was performed using anti-ubiquitin antibodies. The position of the ubiquitinated GST-Siah2 or GST-RNF5 is indicated. Western blot with GST antibodies revealed the amount of ubiquitin ligases used in these reactions (lower panel). (D) In vivo auto-ubiquitnation of Siah2 is inhibited by MEN. 293T cells were co-transfected with Flag-Siah2 with or without HA-tagged ubiquitin. After 24 h, cells were treated with MEN (25 µM) or MG132 (40 µM) for 6 h, or left untreated. Siah2 ubiquitination was analyzed by immunoprecipitation with an anti-Flag antibody (M2) followed by immunoblotting using monoclonal anti-HA antibodies. The same blot was reprobed with anti-Flag antibodies to monitor Siah2 levels. (E) MEN increases PHD3 protein level. MEF cells were treated with indicated concentrations of MEN under normoxia (N) or hypoxia (H; 1% O₂) for 5 h. The DMSO-treated cells were used as a control. The cells were collected, lysed and level of PHD3 determined by Western blot analysis using the indicated antibody. (F) PHD3 is stabilized by menadione treatment. The half-life of PHD3 protein was monitored by cycloheximide (CHX) pulse-chase experiment. MEF cells were treated with MEN (25 µM) or left untreated under hypoxia for 5 h. After 5 h, CHX (20 µg/ml) was added and cells were harvested at the indicated time points and analyzed using the indicated antibodies. Right panel: Densitometric quantification of PHD3 levels, normalized to β-actin levels.