Horseradish peroxidase

IB was carried out as described previously^{19 (link),29 (link)}. Briefly, cells were grown in six-well tissue culture plates in medium supplemented with 10% FBS for 48 h and transfected with a siRNA pool targeting ELF1 or MAZ and an expression vector bearing the HA-tagged ELF1 or the MAZ cDNA for 48 h. Cells were collected and protein isolation was performed using the NE-PER protein extraction kit (Thermo-Fisher). Protein concentration was determined using Bradford Proteins Assay (Bio-Rad). Nuclear extracts were subjected to denaturing SDS-PAGE, transferred to a membrane and proteins were probed with an antibody specific to ELF1 or MAZ followed by a secondary antibody conjugated with the horseradish peroxidase (Advansta). The membranes were then re-probed with the HA antibody (Abcam, ab9110) and subsequently with an HDAC1 antibody (Abcam, ab19845) as a loading control. Images were developed using the ECL-Substrate (Advansta) and captured with ChemiDoc Imaging System (Bio-Rad).

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.