Chemiluminescent substrate

Human liver samples were homogenized with 300 μl lysis buffer containing 10 mM HEPES pH 7.9, 137 mM NaCl, 10% glycerol, 1% NP-40, 1mM PMSF supplemented with protease inhibitor cocktail (Roche, South San Francisco, CA, USA). Total protein concentrations were measured using Bradford method (Thermofisher Scientific, California, USA). MCF7 whole cell lysates prepared with RIPA lysis buffer (Millipore Sigma) were used as a positive control. Capillary Western blot analyses were performed using the Protein Simple Jess system (Biotechne, California, USA) according to manufacturer’s protocol. Briefly, tissue or cell lysates were diluted with 0.1 × sample buffer to concentration of 1 mg/ml. Then 4 parts of diluted samples were combined with 1 part 5 × Fluorescent Master Mix (containing 5 × sample buffer, 5 × fluorescent standard and 200 mM DTT) and heated at 95°C for 5 min. Then the denatured samples, blocking reagent, mouse anti- ESR1 antibodies (D-12 and F-10, at 1:10 dilution, Santa Cruz, California USA), HRP-conjugated anti-mouse secondary antibody (1:20) and chemiluminescent substrate (Biotechne, California, USA) were dispensed into designated wells in an assay plate. A biotinylated ladder provided molecular weight standard for each assay. After plate loading, the separation, electrophoresis and immunodetection steps take place in the fully automated capillary system.