Figure 4
Phosphorylation of S857 is important for the activation of human MIF promoter by SRC-3, CBP and HIF-1α. (A) MCF-7 cells were transfected with the MIF promoter, expression vectors for HIF-1α, wild-type SRC-3 or S857A mutant, either alone or combined as indicated. The luciferase activity was determined as previously described. Shown is the mean ± SD from three experiments performed in duplicate. (B) Cell lysates from MCF-7 cells were used to determine the interaction between endogenous SRC-3 and HIF-1α by co-immunoprecipitation (Co-IP; left panel). HEK293T cells transfected with HIF-1α, alone or together with wild-type SRC-3 or S857A mutant as indicated, were used for Co-IP experiments. Top right panel showed the levels of HA-HIF-1α co-immunoprecipitated with Flag-SRC-3 (middle panel). Bottom panel showed equal expression of HIF-1α. Shown is a representative from three experiments with similar results. Normalized intensity is quantified by ImageJ. (C) MCF-7 cells were transfected with the MIF promoter, expression vectors for HIF-1α, CBP, wild-type SRC-3 or S857A mutant, either alone or combined as indicated. The luciferase activity was determined as previously described. Shown is the mean ± SD from three experiments performed in duplicate. Due to higher number (four) and more total amount of plasmids transfected, less activation by SRC-3 was observed under this particular condition. (D) The MCF-7 cells were first transfected with siRNAs to knockdown the protein indicated. The next day, these cells were transfected as described in C. The luciferase activity was also determined as described in C. (E) The lysates from MCF-7 cells transfected with the indicated siRNAs either alone or in different combination were used for immunoblot to determine the levels of endogenous MIF. β-actin was used as loading controls.
