Tff1 ps2

MCF-7 cells were seeded in 100 mm dishes at a density of 500,000 cells per dish and treated with 0.1% DMSO or 100 pM E2 in the presence or absence of 10 μM LH1092, 10 μM LH1095, or 100 nM CDDO-IM. After 24 hours of treatment, cells were lysed in RIPA buffer containing 1% protease and phosphatase inhibitor. Cell lysates were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membranes were incubated with primary antibodies at 4°C overnight and with secondary antibodies at room temperature for 1 hour. Primary antibodies against PGR (1:500, 8757), c-MYC (1:1000, 5605), TFF1/pS2 (1:1000, 15571), and Cathepsin D (1:1000, 2284) were from Cell Signaling Technology (Danvers, MA). Primary antibodies against HO-1 (1:200, sc-10789) and NQO1 (1:100, sc-271116) were from Santa Cruz Biotechnology (Dallas, TX). β-actin antibody (1:2000, A1978) was from Sigma Aldrich (Saint Louis, MO). Anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling Technology (Danvers, MA). β-actin was used as a loading control.