Western Blot Analysis of Placental Proteins

Western blot analysis was performed as described previously5 (link). Briefly, the placenta was homogenized to obtain protein samples. Then, the proteins (50 μg) were resolved by SDS-PAGE, transferred onto PVDF membranes, and probed with anti-phosphorylation antibodies to Akt (1:1000, Cell Signaling Technology, Inc., USA), mTOR (1:1000, Cell Signaling), p70S6K (1:1000, Bioworld Technology), ERK1/2 (1:1000, Cell Signaling) and antibody of TNF-α (1:500, Bioworld Technology) at 4 °C overnight. The membranes were then incubated with an HRP-labeled secondary antibody and developed using an ECL detection kit (Millipore, MA, USA). Following visualization, the blots were stripped by incubation in stripping buffer (Restore, Pierce) for 5 min, and then incubated with antibodies to Akt (1:1000, Cell Signaling), mTOR (1:1000, Cell Signaling), p70S6K (1:1000, Bioworld Technology), ERK1/2 (1:1000, Cell Signaling) and β-actin (1:1000, Cell Signaling). Western blot bands were scanned and analyzed with the National Institutes of Health Image image analysis software package.