Anti stat6 antibody

After transfection with specific siRNA or none targeting control siRNA (100 nM) cells were lysed in RIPA buffer containing protease inhibitors. 25 μg of total protein was loaded in Lammeli buffer with beta-mecaptoethanol and ran on a 10% Polyacrylamide denaturing gel. The gel was transferred to onto nitrocellulose membrane with wet transfer, blocked for 1 hour in 5% blocking solution (non-fat milk) in TBS-tween and then hybridised overnight with anti-STAT6 antibody (Cell signal, 9362) overnight 1:800 at 4 °C, washed three times in TBS-tween and then incubated with anti-Rabbit Horse Radish Peroxidase (HRP) antibody 1:4000 (Dako, UK) for 1 hour at room temperature, washed and then incubated with ECL substrate (Biorad, UK) and then exposed and developed. Healthy or SSc dermal fibroblast early passage were seeded and then lysed and then probed with anti-STAT6 1:800 or Ten Eleven Translocation-1 (TET1) (Abcam, UK) 1:700 or GAPDH (Abcam, UK) 1:8000. In some experiments healthy dermal fibroblasts where treated with increasing doses of recombinant TGF-β1 and then lysed and then probed for MeCP2 using an MeCP2 specific antibody (Ab2828) and the reprobed for β-actin (Ab8226).