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Figure 3 | BMC Biotechnology

Figure 3

From: New stably transfected bioluminescent cells expressing FLAG epitope-tagged estrogen receptors to study their chromatin recruitment

Figure 3

Compared efficiencies of FLAG-ERα/β immunoprecipitations. A) Soluble chromatin was prepared from HELN-Fα or HELN-Fβ cells stimulated 1 hr with 10 nM of E2, either by using chromatin cross-linked with the same procedure as that used for kinetic ChIP experiments (named "+cross"), or by using uncrosslinked chromatin solubilized in a mild buffer (named "-cross"). Then, 40 μg of ± cross-linked chromatin were subjected to a 10% slab gel electrophoresis before (Inputs) or after (IP) immunoprecipitation with the anti-FLAG antibody. B) HELN-Fα or HELN-Fβ cells were stimulated 1 hr with 10 nM of E2, then, soluble chromatin preparation and immunoprecipitation (with the HC20 anti ERα antibody) were performed by using an identical procedure than that used for ChIP-kinetics experiments. 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 at indicated time were expressed as a percentage of the maximum value obtained in this experiment. Values are mean ± SD of two independent immunoprecipitation assays.

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