Leptin receptor

After treatment with leptin or the control, the cells were harvested in cold PBS, and the pellet was re-suspended in lysis buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol) for 20 min at 4ºC. Next, the lysate was sonicated and centrifuged at 14,000 g for 20 min at 4°C, after which, the supernatant was collected. The protein concentrations were determined using the Bradford assay. A total of 50-100 μg of the protein extract was loaded in each lane, separated on a 10% SDS-PAGE gel, transferred to nitrocellulose membranes and incubated overnight with the following primary antibodies: leptin receptor (1:1000) and CD44 (1:1000) (Santa Cruz Biotechnology, CA, USA); phospho-STAT3 (1:1000), MAPK (1:1000), phospho-MAPK (1:1000), phospho-AKT (1:1000), mTOR (1:1000), phospho-MYPT1 (1:1000), RhoA (1:1000), PARP (1:1000), and Snail (1:1000) (Cell Signaling Technology), Nanog (1:1000) (R&D Systems); and N-cadherin (1:1000), E-cadherin (1:500), Oct-4 (1:1000), Nanog (1:1000), Zeb2 (1:1000), Vimentin (1:1000) (Abcam) and β-actin (1:10000) (Sigma-Aldrich Corp.). Peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibodies (1:3000, Bio-Rad Labs, CA, USA) were applied for one hour at room temperature (RT). The reaction was developed with chemiluminescence using the ECL Western blot analysis system (NEN, Western lightning, Perkin-Elmer).