Western blotting

Whole-cell extracts were prepared with a nuclear extraction TransAM kit (Active Motif, Carlsbad, CA, USA). Nuclear extracts (25–40 µg per lane) were separated on a NuPAGE 10% Bis-Tris Gel (Thermo Fisher Scientific). Electrophoresis was performed at 100 V for 2 hours using NuPAGE® MOPS SDS Running Buffer (Thermo Fisher Scientific). Separated proteins were electrotransferred at 25 V for 25 minutes to a polyvinylidene difluoride membrane using the Trans-Blot semi-dry system (Bio-Rad, Hercules, CA, USA) in Tris/glycine buffer (Bio-Rad). Blots were blocked overnight at 4°C in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.075% Tween-20 (TBS-T), after which they were incubated with mouse anti-PR (AB-52) (1:200 dilution; Santa Cruz Biotechnology Inc., Dallas, TX, USA) or mouse anti-β-actin (1:2000; EMD Millipore) for 2 hours at room temperature. Blots were then washed three times with TBS-T for 10 minutes each, incubated with antimouse IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 hour at room temperature, and washed seven more times with TBS-T for 10 minutes each. Immunoreactive bands were visualized using the ECL plus detection kit (Amersham Pharmacia Biotech, Arlington Heights, IL, USA) using the Bio-Rad ChemiDoc XRS system.