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. 2014 Feb 28;289(16):11219–11229. doi: 10.1074/jbc.M113.535989

FIGURE 5.

FIGURE 5.

SRC-3 coactivated HIF1α in vitro. A, ectopic SRC-3 protein interacted with HIF1α in T24 cells. T24 cells were transiently transfected with FLAG-tagged SRC-3. Co-immunoprecipitation (IP) assay was carried out in cells treated under either normoxia or hypoxia conditions. The precipitant by the IgG antibody was used as a negative control. B, schematic representation of five fragments of SRC-3 representing different domains. C, five fragments of SRC-3 were generated in E. coli BL21(DE3) as GST fusion proteins. The purified GST fused proteins were incubated with HEK293T lysate, which contained the ectopic expression of HA-HIF1α. Western blot (WB) was performed to detect the pulled down HIF1α by HA antibody. D–H, luciferase activity assay: D, in the HRE-Luc-transfected 293T cells co-transfected with SRC-3 expression plasmid, with or without DFO treatment, ***, p < 0.001, compared empty vector transfection group; ###, p < 0.001 compared with the SRC-3 transfection group; E, in HRE-Luc 293T cells with co-transfection of SRC-3 and HIF1α expression plasmids with the indicated amount of plasmids, ***, p < 0.001; F, in the PGK1-reporter 293T cells co-transfected with the SRC-3 expression plasmid, with or without DFO treatment, ***, p < 0.001, compared with the empty vector transfection group; ###, p < 0.001, compared with the SRC-3 transfection group; G, in the PGK1-reporter 293T cells with co-transfection of SRC-3 and HIF1α expression plasmids. PGK1-Luc, wild type PGK1 reporter; ΔPGK1-Luc, PGK1 reporter without HRE; followed by transfection with or without HIF1α. H, in HRE-Luc T24 cells transduced with control (−) or SRC-3 shRNA (+), with or without HIF1α overexpression, ***, p < 0.001, compared with the empty vector transfection group; ###, p < 0.001, compared with the group with SRC-3 knockdown and HIF1α overexpression. I, presence of HIF1α and SRC-3 on the endogenous glut1 promoter and pgk1 promoter from the pgk1-luc reporter detected by the ChIP assay. Parental T24 cells (left panel) and T24 wells transfected with the pgk1-luc reporter plasmid (right) were incubated under normoxia (N) or hypoxia (H), respectively. The band intensity was shown under each blot, which was quantified by ImageJ software. The precipitant by IgG antibody was used as a negative control. RLU, relative light units.