Imagelab software ver 6

Passage 1 hESCs were harvested and lysed with cell lysis buffer (Merck KGaA, Darmstadt, Germany) with Protease Inhibitor Mixture (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitors (Nacalai Tesque). The cell lysates were separated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membrane was blocked by incubation with skim milk in 0.1% Tris-buffered saline with Tween 20 (TBST) for 90 min. Subsequently, the membrane was incubated overnight at 4 °C with primary antibodies. The primary antibodies used were anti-ERα (H-184) and -GAPDH (FL-335) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. The following day, the membrane was washed with TBST three times for 5 min each time. Subsequently, the membranes were incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Sigma Aldrich) for 1 h at 37 °C, followed by treatment with Clarity Western ECL Substrate detection kits (Bio-Rad), and subjected to a ChemiDoc XRS Touch Imaging System (Bio-Rad). The intensities of the bands were quantified with Image Lab software (ver. 6.0; Bio-Rad) and were normalized against the bands obtained for GAPDH. MCF-7 cells were used as a positive control. All the antibodies used are listed in Supplemental Table S5.