Fig. 5.
Effects of ligands on protein levels and ERE interactions of ERα and ERαEBD with or without GFP. A. Transiently transfected HeLa cells for 24h were incubated with fresh media supplemented with or without 10−9 M E2, 10−7 M 4HT or ICI for 1h. Cells were then collected, washed, re-suspended in 1 ml PBS and divided into two equal portions. One portion of collected cells was pelleted and subjected to protein extraction using a high salt extraction buffer (HSB). The remaining pellet was subjected to 1x Laemmli Buffer (LB) to extract insoluble receptor aggregates. The other portion of the suspended cells was pelleted and the pellet was suspended with LB to extract both soluble and insoluble proteins for total cell lysate (TCL). Ten μg total protein was subjected to SDS 10–18%PAGE. Proteins with (G) or without GFP were probed with a receptor specific antibody. All experiments were replicated at least two independent times. B. Chromatin Immunoprecipitation (ChIP) of transiently transfected HeLa cells. Cells co-transfected with expression vector expressing an ERα cDNA and the reporter vector bearing the TATA box promoter with single ERE for 24h were treated without (NL) or with 10−9 M E2, 10−7 4HT or ICI for 1h. Cells were then subjected to ChIP using Flag-M2 antibody conjugated agarose beads. A 366 bp PCR fragment indicates the ER-ERE interactions. Experiments were replicated at least three independent times. C. The in situ ERE binding competition assay. HeLa cells were co-transfected with 125 ng the TATA box promoter with one ERE that drives the expression of the firefly luciferase cDNA as the reporter enzyme and 300 ng expression plasmid bearing the designer transcription factor, PPVV, without (0 ng ER) or with 75, 150 or 300 ng expression vector bearing an ER cDNA with or without GFP. Cells were then grown in the medium supplemented without (NL) or with 10−9 M E2, 10−7 M 4HT or ICI for 24h. Normalized luciferase activity is presented as percent change compared to the control (PPVV, 0 ng ER) without ligand, which was set to 100. Graph represents the mean of three independent experiments performed in duplicate; SEM, which was less than 15% of the mean, is not shown for simplicity. D. Transfected HeLa cells with an expression vector bearing none (V) or an ERβ cDNA were treated without (NL) or with 10−9 M E2, 10−7 M 4HT or ICI for 1h. Cells were collected, pelleted and subjected to protein extraction using HSB. Ten μg total protein was subjected to SDS 10–18%PAGE. Proteins were probed with a receptor specific antibody. The image is from an experiment that was repeated at least three independent times. E. ChIP assays for in situ interactions of ERβ and ERβEBD with ERE in HeLa cells were carried out using the M2-Flag antibody conjugated agarose beads as described for ERα proteins. A representative image from an experiment repeated three independent times is shown. F. Transient transfections of HeLa cells for the assessment of the binding of ERβ proteins with (G) or without to ERE using the in situ ERE binding competition assay are accomplished as described for ERα. The mean of three independent experiments performed in duplicate without the SEM, which was less than 15% of the mean, is shown.