Samples were thawed, resuspended in Laemmli Buffer (20% Glycerol, 4% SDS in 100 mM Tris Buffer, pH 6.8) and lysed in a Tissue homogenizer (Precellys Evolution, Bertin Instruments). BC Microstructure lysates were recovered, sedimented to remove cell debris, sonicated and stored at − 80 °C until use.
Protein quantification was performed in a Nanodrop ND-2000C (Thermo Scientific). Proteins were denatured and loaded in a electrophoresis gel (NuPAGE 4–12% Bis-Tris Gel) under reducing conditions for 50 min (200 V) and then electrophoretically transferred using a wet transfer system (Bio-Rad, 30 V, 18 h, 4 °C) into nitrocellulose membranes. Membranes were blocked for 1 h in TBS with 0.1% (w/v) Tween 20, 5% (w/v) non-fat dried milk and further incubated with the primary antibodies (Mouse anti-Human ERα, 1D5 Clone, Dako, final dilution 1:500; Rabbit anti-β tubulin, H-235, SC-9104, SantaCruz, final dilution 1:1000, used as loading control) and respective secondary HRP-conjugated secondary antibodies (Sheep anti Mouse IgG NA931; Donkey anti Rabbit IgG NA934; GE Healthcare, final dilution 1:20000). Membranes were developed using Amersham ECL Select Western Blot Detection Reagent (GE Healthcare) and visualized using a ChemiDoc System (BioRad).
Protein quantification was performed in a Nanodrop ND-2000C (Thermo Scientific). Proteins were denatured and loaded in a electrophoresis gel (NuPAGE 4–12% Bis-Tris Gel) under reducing conditions for 50 min (200 V) and then electrophoretically transferred using a wet transfer system (Bio-Rad, 30 V, 18 h, 4 °C) into nitrocellulose membranes. Membranes were blocked for 1 h in TBS with 0.1% (w/v) Tween 20, 5% (w/v) non-fat dried milk and further incubated with the primary antibodies (Mouse anti-Human ERα, 1D5 Clone, Dako, final dilution 1:500; Rabbit anti-β tubulin, H-235, SC-9104, SantaCruz, final dilution 1:1000, used as loading control) and respective secondary HRP-conjugated secondary antibodies (Sheep anti Mouse IgG NA931; Donkey anti Rabbit IgG NA934; GE Healthcare, final dilution 1:20000). Membranes were developed using Amersham ECL Select Western Blot Detection Reagent (GE Healthcare) and visualized using a ChemiDoc System (BioRad).