Fvii antibody

Three sets of breast cancer tissue microarray (TMA) slides were obtained from Pantomics (Richmond, CA, USA) that constituted of duplicate cores for a total of 209 malignant breast tumors (BRC1501-3). Immunohistochemistry (IHC) staining was performed as described before [32 (link), 33 (link)]. Primary antibody incubation was carried out with rabbit polyclonal coagulation factor VII (FVII) antibody at 1:200 dilution (Novus Biologicals, Littleton, CO, USA) and mouse monoclonal AR antibody at 1:75 dilution (Dako, Carpinteria, CA, USA). Tumors with ≥10% nuclear-stained cells were considered positive for AR as previously published [8 (link)]. Since FVII has a cytoplasmic staining pattern, a semi-quantitaive scoring system that has been previously used for other cytoplasmic proteins was adapted to score FVII [34 (link)]. In this respect, FVII staining was scored based on the intensity of cytoplasmic staining as follows: no staining (score 0), weak staining (score 1), moderate staining (score 2), and intense staining (score 3).

Western blot analysis was carried out with rabbit polyclonal FVII antibody (Novus Biologicals) at 1:1000 dilution using 50 µg of T-47D cell lysate from each DHT-treated and control experiments. Membrane was stripped and immunoblotting with rabbit α-tubulin antibody (Abcam, Cambridge, UK) was applied to assess loading. To extract protein from the conditioned media, cell lines were cultured for 48 hours in serum-free media containing either DHT at 100 nM or control vehicle followed by concentration using Amicon Ultra-15 (3K) centrifugal filters (Millipore, Billerica, MA, USA). A total of 100 µg from each conditioned medium was precipitated and used for immunoblotting. Protein concentrations were measured using the BCA Protein Assay Kit (Fisher scientific). Immunoblot imaging and analysis of band densities were performed using ChemiDoc XRS system and Image Lab software, respectively (Bio-Rad, Hercules, CA, USA). Western blots were performed in three replicates and the average fold change was shown.