Sc 101058

Protein was prepared in NuPAGE Sample Buffer and Reducing Agent (Life Technologies, Carlsbad, CA, USA) using 10 μg (estrogen-related blots), 65 μg (V5 blot, T47D-ELF5-isoform 2-V5) or 25 μg (V5 blots, all other lines) per lane. Samples were separated on precast 15-well 4–12 % Bis-Tris (estrogen-related blots) or 10-well 10 % Bis-Tris (V5 blots) polyacrylamide gels (Life Technologies), transferred to polyvinylidene fluoride membrane, blocked in 5 % skim milk, and incubated overnight at 4 °C in primary antibody. Secondary horseradish peroxidase–conjugated antibody was added 1:2000 in 5 % skim milk (anti-mouse, NA931V, anti-rabbit, NA934V; GE Healthcare Life Sciences, Little Chalfont, UK). Proteins were detected using enhanced chemiluminescence solution (Western Lightning Plus; PerkinElmer, Waltham, MA, USA) and x-ray film (Fujifilm, Tokyo, Japan). Primary antibodies used were anti-V5 (sc-58052, 1:500–1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-transducin-like enhancer of split 1 (anti-TLE1) (ab183742, 1:1000; Abcam, Cambridge, UK), anti-ERα (sc-8005, 1:1000; Santa Cruz Biotechnology), anti-Forkhead box A1 (anti-FOXA1) (sc-101058, 1:1000, Santa Cruz Biotechnology), and anti-β-actin (AC-15, 1:20,000; Sigma-Aldrich, St. Louis, MO, USA).