β hcg

Mouse monoclonal antibodies against VEGF, factor VIII, β-hCG, hCG receptor, and HIF-1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A rabbit monoclonal antibody against CD31 was obtained from Bioworld (Dublin, OH). Cellular proteins were isolated after rinsing cells with ice-cold phosphate-buffered saline (PBS; pH 7.4) and lysing them on ice with a protein-extraction reagent. The proteins were then separated on an 8 % sodium dodecyl sulphide-Tris polyacrylamide gel. Transfer to a polyvinylidene fluoride membrane was performed at 0.27 mA for 2 h. The membranes were blocked overnight with 1× Tris-buffered saline containing 0.1 % Tween 20 and 5 % skim milk, followed by incubation with primary antibody (1:100) for 1 h and a horseradish peroxidase-conjugated secondary antibody (1:1000) for an additional 1 h at room temperature. Immunocomplexes were visualized by electrochemiluminescence. Protein expression was semi-quantified using a Tiannen imager and analysis system (Shanghai, China). Protein contents in the three independent replicates were respectively quantified and presented as mean ± SD.

Antibodies used for all the experiments were as follows: for immunoflourescence, β-hCG (ab9582, Abcam), Vimentin (5144 S, Cell signaling technology), E-cadherin (3195 S, Cell signaling technology), P-cadherin (sc-7893, Santa Cruz Biotechnology), BRCA1 (9010 S, Cell signaling technology) primary antibodies were followed by secondary antibodies conjugated with FITC (35552, Cell Signaling Technology). For immunoblotting, Vimentin (5144 S, Cell signaling technology), E-cadherin (3195 S, Cell signaling technology), β-actin (sc-47778, Santa Cruz Biotechnology). For immunoprecipitation: β-hCG (sc-271062, Santa Cruz Biotechnology), TGF beta Receptor II (ab78419, Abcam), normal IgG (sc-2025, Santa Cruz Biotechnology). ChIP: BRCA1 (A301-377 A, Bethyl laboratories). Immunohistochemical analysis: β-hCG (AM305-5M, Biogenex), BRCA1 (AR345-5 R, Biogenex).