4.7. Gene Knock-Down with siRNA and Western Blotting

Cells were seeded and transfected after 24 h using Lipofectamine 2000 according to the producers’ protocol. The siRNA used in this experiment was designed to interfere with ERα36 mRNA. The following sequences were chosen: sense 5′-AUGCCAAUAGGUACUGAA-3′ and antisense 5′-UUCAGTACCUAUUGGCAU-3′. We confirmed gene silencing in the MCF7 cell line by Western blotting. Cell lysate was prepared using RIPA buffer (Sigma Aldrich, Saint Louis, MO, USA), while protein concentration was measured by a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated using 12% polyacrylamide TGX gels (Bio-Rad, Hercules, CA, USA) and transferred onto the PVDF membrane by semi-dry transfer (Bio-Rad, Hercules, CA, USA). For detection, we used primary rabbit anti-ERα36 antibody (Cell Applications Inc. San Diego, CA, USA; dilution 1:500), mouse anti-ERα66 antibody (SantaCruz, Dallas, TA, USA; dilution 1:1000), and anti-β-actin antibody (Sigma Aldrich, Saint Louis, MO, USA; dilution 1:10,000). Appropriate, secondary anti-rabbit and anti-mouse HRP-conjugated antibodies were used (Sigma Aldrich, Saint Louis, MO, USA; dilution 1:100,000).

Analysis of ERα36 levels in frozen breast cancer samples with Western blotting was performed according to the protocol described above, apart from the homogenization step, where frozen tumor samples were cut and suspended in RIPA buffer (Sigma Aldrich, Saint Louis, MO, USA); then, tissue was minced using a sterile scalpel and centrifuged at 10,000× g for 10 min in 4 °C. Homogenization was followed by the measurement of protein concentration by a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA).