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GalDBD/HIF-1a 1-245

GalDBD/HLF 1-265

GalDBD

Fig. 3. Reciprocal two-hybrid assay confirming that Ref-1 specifically interacts with the N-terminal region of HLF but not with HIF-la. COS-1 cells were cotransfected with 300 ng of Gal4-luciferase reporter G5Elb-Luc, 50 ng of expression vector chimeric for the DNA-binding domain of Gal4 and HLF 1-265 or HIF-la 1-245, and 50 ng of VP16/Ref-l or VP16 as indicated. After 40 hr cells were assayed for luciferase activity as outlined in Fig. 2. Results are from two independent experiments performed in triplicate and are represented as fold induction relative to reporter gene activity of nonchimeric GalDBD with VP16. Error bars represent the standard deviation.

Ref-1 was fused to the activation domain of VP 16 and then assayed for interaction with a GalDBD chimera containing HLF. As in Fig. 2, coexpression of Ref-1 (VP16/Ref-l) and HLF (GalDBD/HLF 1-265) fusion proteins resulted in an increase in reporter gene activity when compared with coexpression of VP16/Ref-l and GalDBD alone, further confirming that Ref-1 interacts with the N-terminus of HLF. We have previously shown that whereas HIF-la can recognize the same DNA response element as HLF, its DNA-binding activity is not regulated by Ref-1,8 This is due to the basic DNA-binding region of HIF-la containing a serine rather than a cysteine at the critical position 28 (see Fig. 1). In agreement with the DNA-binding experiments, coexpression of VP16/Ref-l with an N-terminal HIF-la fusion (GalDBD/HIF- la 1-245) resulted in no increase in reporter gene activity.

We believe the major advantages of analyzing redox protein interactions with the mammalian two-hybrid system are that (1) it is a cell-based in vivo interaction assay, which is more physiologically relevant than an in vitro interaction assay; and (2) it is often more sensitive than other assays such as coimmunoprecipitation, this being important because most redox interactions tend to be weak and transient in nature. Although other methods to observe transient interactions between redox proteins exist, for example, solution nuclear magnetic resonance analysis of the interaction between thioredoxin and Ref-1,13 the mammalian two-hybrid approach is by comparison simple, quick, and inexpensive, and requires no specialized expertise.

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