Sc 542

Cells were treated with 2 mg/mL D. candidum for 48 and 72 hours. Western blot was performed as previously described.15 The cells were collected in ice-cold phosphate-buffered saline and lysed in ice-cold whole cell extraction buffer containing 25 mM β-glycerophosphate (pH 7.3), 5 mM ethylenediaminetetraacetic acid, 2 mM ethylene glycol tetraacetic acid, 5 mM β-mercaptoethanol, 1% Triton X-100, 0.1 M NaCl, and a protease inhibitor mixture (Roche Applied Science, Penzberg, Germany). The protein concentration cell lysates were determined by using the Bradford method and boiled in sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris, pH 6.8; 100 mM dithiothreitol; 2% SDS; 0.1% bromophenol blue; and 10% glycerol). The proteins were separated on 8%–10% SDS polyacrylamide gel and electrotransferred to polyvinylidene fluoride membrane. After blocking with 3% bovine serum albumin-Tris-Hcl buffer saline Tween, primary antibodies (PGR, MILLIPORE 2437345; ELF5, Abcam ab136119; GATA3, Abcam ab11693; ERα, Santa Cruz Biotechnology sc-542; p53, Abcam ab26; Ki67, Abcam ab15580) were detected using horseradish peroxidase-conjugated antirabbit antibodies and visualized on Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology, Shanghai, People’s Republic of China). β-Actin (ZSGB-BIO TA-09, Beijing, People’s Republic of China) was also tested to confirm equal loading.