Figure 4

HRG-induced expression of the candidate transcription factors. (A) MCF-7 cells were treated with 10 nM HRG or EGF. Cell lysates were assayed for expression of EGR4 and c-FOS proteins using Western blot analysis. A representative figure of two independent experiments is shown. (B-D) HRG-induced expression of mRNA and protein associated with the AP-1 protein. HRG-treated cell lysates were immunoprecipitated using c-FOS antibody and subjected to Western blot analysis. Blots were then probed using an antibody against c-JUN or FRA-1. A representative figure of two independent experiments is shown. Mean values of quantified bands are shown with bars ± range (B). HRG-induced mRNA expression of c-JUN obtained by qRT-PCR (upper) and GeneChip (lower graph) analyses (C). HRG-treated MCF-7 cell lysates were immunoprecipitated using c-FOS, FRA-1 (D) or FHL2 (E) antibody. Immuno-complexes were detected using an antibody against c-FOS, FRA-1 or FHL2. The data are representative of two independent experiments. Mean values of quantified bands are shown with bars ± range. (F) EMSA analysis of FHL2 DNA-binding ability. ³²P-labeled -318 TRE oligonucleotide probe, the sequence of which is present within the FOSL-1 promoter region, was incubated with HRG-treated cellular nuclear extract. For the competition assay, nuclear extracts were incubated with a 50-fold molar excess of unlabeled probe prior to use in the EMSA experiment. The -318 TRE sequence incubated with nuclear extract was observed in the control lanes and 'shift' bands indicate protein-nucleic acid complex formation. Supershift experiments of the complexes formed with the consensus -318 TRE sequence using FHL2 antibody.