H 190

Isolated protein was applied to a Bis-Tris NuPAGE 4%−12% gel (Novex by Life Technologies) for protein separation. Protein was wet transferred to a polyvinylidene difluoride membrane and blocked in 5% blotting grade nonfat milk diluted in phosphate-buffered saline (PBS) with 0.1% Tween for at least one hour. Membranes were incubated with primary antibody (PGR Santa Cruz H-190 (1:400), ACTIN Santa Cruz I-19 (1:10,000), myc-tag Origene TA100010 (1:1000), CCND1 Thermo Fisher RB-9041-P0 (1:200), mTORC1 Cell Signaling 2983 (1:1000), pmTORC1 Cell Signaling 2971 (1:1000), COX1 Cayman Chemical 160110 (1:200), COX2 Cell Signaling 12282 (1:1000), ERK Cell Signaling 9102 (1:1000), pERK Cell Signaling 4370 (1:1000)) overnight. Membranes were washed and incubated with secondary antibody (anti-rabbit peroxidase (1:4000), anti-mouse peroxidase (1:5000), and anti-goat peroxidase (1:4000) according to primary antibody requirements) in 5% milk diluted in PBS with Tween. The Amersham ECL Western blotting system was utilized to develop peroxidase labeled protein according to manufacturer’s instructions (GE Healthcare).

Ishikawa PR-B cells, in steroid free media, were treated with indicated concentrations of purified compounds for 24 hours. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors and the protein concentration was determined via BCA. Twenty-five micrograms of protein was separated on SDS-PAGE cells by electrophoresis and transferred to nitrocellulose membranes.^{36 (link)} Membranes were blocked for one hour in 5% milk and probed overnight for progesterone receptor (diluted 1:500, H-190, Santa Cruz Biotechnology) or β actin (diluted 1:1000, A2066, Sigma Aldrich, St. Louis MO). The next day, membranes were washed with TBS-T, incubated with anti-rabbit secondary antibody and developed with SuperSignal West Femto Substrate (34095, Thermo Scientific, Rockford IL). Images were captured with a FluorChem C (Protein Simple, San Jose, CA).