Figure 4

Recruitment of ERα/β to the promoter of the luciferase transgene in HELN-Fα/β cells. Kinetic ChIP experiments were performed using the anti-FLAG antibody. HELN-Fα (A) or HELN-Fβ (B) cells were cultured in 3% dextran-charcoal treated FCS. Twenty four hours before experiment, they were deprived of serum and subsequently treated for 2 hr with 2.5 μM α-amanitin, and then with 10 nM E2. Cells were cross-linked at indicated times. Soluble chromatin was prepared on sampled cells at indicated times as described in material and methods. Real time PCR quantification of either IP chromatin or input was performed at each incubation time. Amplified signals from IP chromatin were calculated as the percentage of amplified input signals obtained during the same amplification. Corresponding values plotted for one curve were expressed as the percentage of the maximum value of % input (% input max) obtained for that curve and for one IP. One ChIP kinetic curve shown is representative of at least two independent experiments and values are mean ± SD of two independent immunoprecipitation assays using the same preparation of chromatin. The % input max average value (corresponding to the two IPs) of one curve may fluctuate among different independent experiments corresponding to the same kinetic. The corresponding amplitudes of variation are: in A (HELN-Fα) Imin = 2.1, Imax = 4.3; in B (HELNβ) Imin = 1.4 Imax = 3.5. Flag-ERα (A) or Flag-ERβ (B) protein levels were analyzed by Western blot experiments. Cell treatments for ChiP assays and Western blot experiments were identical. Extracts were prepared at indicated times, and Western blotted with antibodies for FLAG (F3165) or actin.