FIG. 6.
HU reduces Rfx1 DNA binding. A. ChIP of the Rfx1 promoter. Cross-linked chromatin was precipitated with either anti-Rfx1 polyclonal antibodies (+; lanes 2 and 4) or with control preimmune rabbit serum (−; lanes 1 and 3) in the presence or absence of 1 mM HU. The amount of the Rfx1 promoter in the input and precipitated (IP) fractions was determined by PCR. B. EMSA with extract from MCF-7 cells grown in medium containing charcoal-filtered serum with or without 1 mM HU by use of the x-box sequence as probe. The expected complexes are marked a and b. C. RT-PCR on RNA extracted from MCF-7 cells treated as described for panel B using Rfx1-specific primers or β-actin-specific primers as controls. D. Quantification of Rfx1 RT-PCR band intensity by use of Exbam 3.0.4 software (Pixlock). Error bars represent three independent experiments. E. Western blot of the extracts described in for panel B. F. RT-PCR performed using total RNA from MCF-7 cells irradiated with 20 J/m2 UV at different times postirradiation by use of Rfx1- and β-actin-specific primers.