Monoclonal mouse anti hsc70 antibody sc 7298

Benzo[a]pyrene (B[a]P), 7,12-Dimethylbenz[a]anthracene (DMBA), α-naphthoflavone (α-NF), cytochalasin B, insulin, 2-deoxyglucose and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). N-(Diaminomethylene)-4-isopropyl-3-(methylsulfonyl)benzamide (Cariporide) was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Hoechst 33342 was purchased from Life Technologies (Les Ulis, France). All these products were used as a stock solution in DMSO; the final concentration of this vehicle in the culture medium was <0.00005% (v/v), and control cultures received the same concentration of vehicle as treated cultures. [³H]-2-deoxyglucose was from PerkinElmer (Boston and Waltham, USA). Monoclonal mouse anti-HSC70 antibody (sc-7298) and monoclonal mouse anti-actin antibody (sc-8432) were purchased from Santa Cruz Biotechnology. Monoclonal mouse anti-E-Cadherin antibody (610404) was purchased from BD Biosciences (Le Pont de Claix, France), and monoclonal mouse anti-vimentin antibody (M0725) and secondary antibodies conjugated with horseradish peroxidase, from DAKO (Les Ulis, France).

Sample preparation and Western blotting were performed using standard techniques. Nitrocellulose membranes (0.45 µm Amersham Protran; 10600002; GE Healthcare, Buckinghamshire, UK) were incubated with the following antibodies at the dilution indicated: monoclonal rabbit anti-phospho-STAT5 (Y694) antibody (9351; 1:1000; Cell Signaling Technologies, Danvers, MA, USA), monoclonal mouse anti-STAT5 antibody (610191; 1:1000; BD Biosciences Pharmingen, San Jose, CA, USA), polyclonal rabbit anti-PARP antibody (9542; 1:1000; Cell Signaling Technologies), monoclonal mouse anti-HSC70 antibody (SC-7298, 1:5000, Santa Cruz, St. Louis, MO, USA).