Figure 1.

VDU2, but not VDU1, interacts with HIF-1α. (A) GST pull-down assay for mapping the HIF-1α–VDU2 interaction. In vitro-translated VDU2, VDU2 1–467, VDU2 467–913, VDU2 269–390 and VDU1 proteins were incubated with GST–tHIF-1α (530–826) and the bound proteins were eluted and analysed using SDS–polyacrylamide gel electrophoresis. Equal loading of GST fusion proteins is demonstrated in the lower panel. (B) In vivo co-immunoprecipitation (Co-IP) of HIF-1α with GFP–VDU2. Lysates prepared from COS-7 cells transfected with pCEP4–HIF-1α and pEGFP–C3 or pEGFP–C3–VDU2 were subjected to IP with anti-HIF-1α antibody followed by anti-GFP immunoblotting (IB). (C) In vivo Co-IP of HIF-1α with GFP–VDU1 or GFP–VDU2M-C154A. Lysates prepared from COS-7 cells transfected with pCEP4–HIF-1α plus pEGFP–C3, pEGFP–C3–VDU1 or pEGFP–C3–VDU2M-C154A were subjected to IP with anti-HIF-1α antibody followed by anti-GFP IB. (D) Interaction between endogenous HIF-1α and VDU2. The top panel shows a western blot of control IP with the pre-immune serum (lanes 1,2) and IP with VDU2 antibody (lanes 3,4) from HeLa cells untreated (lanes 1,3) or treated with CoCl₂ (lanes 2,4). The middle and bottom panels show blots of cell extracts by anti-HIF-1α and anti-β-actin antibody, respectively.