Tgfβr2

Protein extraction and Western blot analysis were performed in MCF-7 cells induced or non-induced with TGFβ1 and EGF. MCF-7 cells (5 × 10⁵) were plated in 6 wells plate followed by incubation with TGFβ1 and/or EGF at several concentrations (0, 5 and 10 ng/ml for TGFβ1 and 0 or 20ng/ml for EGF). After 72 h of treatment cells were lysed in sample buffer (62.76 mMTris– HCl pH 6.8, 5% 2-mercaptoethanol, 2% SDS, 10% glycerol, 0.5%bromophenol blue, and 100 mM dithiothreitol). Proteins (30 μg) were separated by SDS-PAGE (10%) in a Mini Protean II cell (Bio-Rad, Hercules, CA) and were transferred to a nitrocellulose membrane (80 V at room temperature for 30 min). Blots were treated with blocking solution (PBS TWIN 0.5% non-fat milk) for 1 h at room temperature and then reacted with primary antibody against VIM, Slug, Bcl-2, caspase 9, multi-CK, TGFβ R2 (Abcam, Cambridge) and EGFR (Santa Cruz Biotechnology, CA) at 1:1000 dilution overnight at 4°C. Then, membranes were washed and reincubated for 1h with a horseradish peroxidase-conjugated anti-IgG (Abcam, Cambridge) diluted at 1:1000. Protein–antibody complexes were visualized by enhanced chemiluminescence (ECL Prime western blotting Detection Reagent, GE Healthcare, Buckinghamshire) and using a densitrometric analysis system (J1·47t image processing system, National Institute of Health, USA).

CD4⁺YFP⁺ T_reg cells and CD4⁺YFP⁻ conventional T cells were sorted from Foxp3^Cre and Foxp3^CreLkb1^f/f mice and treated as indicated, and then cells were lysed and western blot was carried out as previously described55 (link) with antibodies to Lkb1 (D60C5, Cell Signalling Technology), phosphorylated AMPK (40H9, Cell Signalling Technology), IKKβ (2C8, Cell Signalling Technology), phosphorylated IKKα/β (16A6, Cell Signalling Technology), phosphorylated IκBα (14D4, Cell Signalling Technology), phospho-NF-κB p65 (93H1, Cell Signalling Technology), NF-κB p65 (D14E12, Cell Signalling Technology), STAT4 (C46B10, Cell Signalling Technology), AMPKα (D63G4, Cell Signalling Technology), phospho-Acetyl-CoA Carboxylase (D7D11, Cell Signalling Technology), phospho-STAT4 (Abcam), TGF-βR2 (Abcam) and GAPDH (D16H11, Cell Signalling Technology).