Ab106533

WB was performed as described previously^{4 (link)}. In brief, cells grown in six-well tissue culture plates in medium supplemented with 10% CD-FBS to reduce the endogenous E2 concentration for 48 h were transfected with an siRNA specific for CXXC5 or a non-target control siRNA (AllStar, CtS) in the absence (EtOH, 0.01%) or the presence of E2 (10⁻⁸ M) for 48 h. At the termination, cells were collected and the nuclear content was isolated using NE-PER protein extraction kit (Thermo Fisher). Protein concentration was measured with Bradford Protein Assay (Bio-Rad). Nuclear extracts (50 μg) were then subjected to 12% SDS-PAGE. Proteins were probed with an antibody specific to CXXC5 (ab106533, Abcam) or PARP1 (9542; Cell Signaling Tech.) followed by a secondary antibody conjugated with horseradish peroxidase (Advansta Inc., San Jose, CA, USA). The HDAC1 antibody (Abcam, ab19845) was used for monitoring the levels of HDAC1, which was used as the loading control. Proteins were visualized with ECL (Advansta) and images were captured with ChemiDoc Imaging System (Bio-Rad). PageRuler Prestained Protein Ladder (Thermo-Fisher) was used as a molecular weight marker.

The growth and maintenance of E2 responsive and ERα-synthesizing MCF7 and T47D cells were described previously^{4 (link),64 (link)}. In all experiments, media were changed every third day when appropriate.
Restriction and DNA modifying enzymes were obtained from New England Bio-Labs (Beverly, MA, USA) or Thermo-Fischer Sci. (Waltham, MA, USA). 17β-estradiol (E2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The complete antagonist of estrogen receptor, Imperial Chemical Industries 182780, (ICI) was obtained from Tocris Biosciences (Ellisville, IL, USA). The antibody for Poly(ADP-ribose) polymerase 1 (PARP1; 9542) was obtained from Cell Signaling Technology (Beverly, MA, USA). The antibodies for CXXC5 (ab106533) and for HDAC1 (ab19845) were purchased from Abcam Inc. (Cambridge, MA, USA). Secondary antibodies conjugated with horseradish peroxidase were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). siRNAs (FlexiTube siRNA) for CXXC5 were purchased from Qiagen Inc. (Düsseldorf, Germany). Camptothecin (13637) was purchased from Cell Signaling Technology.

WB was carried out as described previously46 (link)47 (link)49 . In brief, MCF7 cells grown in six-well tissue culture plates in medium supplemented with CD-FBS for 48 h were treated without (EtOH, 0.01%) or with 10⁻⁹ M E2 and/or 10⁻⁷ ICI for 3, 6 or 24 h. At the termination, cells were collected and protein isolation was performed using NE-PER protein extraction kit (Thermo-Fisher). Protein content in extracts was measured with Bradford Protein Assay (Bio-Rad). Nuclear extracts (25 μg or 100 μg) were then subjected to SDS 10%-PAGE. Proteins were probed with an antibody specific to CXXC5 (ab106533, Abcam) or Flag (Sigma-Aldrich) followed by a secondary antibody conjugated with the horseradish peroxidase (Santa Cruz). Protein images were developed using the ECL-Plus Western Blotting kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and captured with ChemiDoc™ Imaging System (Bio-Rad). Precision Plus Protein™ Dual Color Standards (Bio-Rad) was used as molecular marker in WB. The quantification of images was carried out using ImageJ image processing program (https://imagej.nih.gov/ij/).