FIGURE 4.

FHL1 disrupts binding of HIF-1α to p300/CBP co-activators. A, FHL1 interacts with p300 and CBP. 293T cells transfected with V5-FHL1 were lysed and immunoprecipitated with a control IgG or V5 antibody. Immunoprecipitates were probed with p300, CBP, and Myc antibodies. B, mammalian two-hybrid assay of HIF-1α TAD-p300 interaction. C, FHL1 inhibits interaction between the HIF-1α C-TAD and p300 CH1 domain. 293 cells were co-transfected with Gal4-HIF-1α(786–826) and VP16-CH1 expression vectors; pG5-E1b-Luc and pSV-RL reporter plasmids; and EV, FHL1, or FHL2 vector. 24 h post-transfection, the cells were exposed to an additional 24 h of hypoxic or nonhypoxic conditions. The cells were harvested, and the ratio of firefly:Renilla luciferase was determined. The results are shown as the means ± S.D. *, p < 0.01 compared with EV. D, FHL1 disrupts HIF-1α-p300 interaction. 293T cells were co-transfected with vector encoding FLAG-tagged double mutant (P402A/P564A) HIF-1α (DM) and either EV, FHL1, or FHL2 vector as indicated. 24 h post-transfection, the cells were left untreated or treated with 100 μm DFX for 6 h, then lysed, and subjected to IP with anti-FLAG antibodies. Immunoprecipitates and cell lysates were subjected to WB with antibodies against p300, FLAG, or Myc. E, FHL1 disrupts HIF-1α-CBP interaction. 293T cells were co-transfected with FLAG-HIF-1α(DM) vector and either EV or vector encoding FHL1 or FHL2 as indicated. 24 h post-transfection, the cells were left untreated or treated with 100 μm DFX for 6 h, then lysed, and subjected to IP with anti-FLAG antibodies. Immunoprecipitates and cell lysates were subjected to WB with antibodies against CBP, FLAG, V5, or Myc.