Total p38 mapk

Amniotic sac and placenta samples were lysed in a RIPA lysis buffer with freshly added protease and phosphatase inhibitors (0.01%). The insoluble material was removed by centrifugation at 10,000 rpm for 20 min at 4°C. The concentration of protein in each tissue lysate was determined by using the BCA protein assay kit (Pierce BCA Protein Assay Kit, Thermo Scientific). The same amount of protein (30 µg) from each sample was loaded onto a 10% SDS-PAGE gel and electrophoresed at 120 V. The resolved proteins were transferred to a PVDF membrane using the iBlot transfer apparatus (Bio-Rad Laboratories). The membranes were blocked in Tris-Buffered Saline (TBS) containing 0.1% Tween 20 (TBS-T) and 5% skim milk for 2h at room temperature. Blots were incubated separately with total p38MAPK (Cell Signaling, Danvers, MA, USA, #9212), phosphorylated (P)-p38MAPK (Cell Signaling, #9211S), or β-actin (Sigma-Aldrich, #A5441) specific primary antibody at 4°C and shaken overnight. Blots were washed three times with TBS-T and incubated with appropriate peroxidase-conjugated IgG secondary antibody for 1h at RT. All blots were developed using chemiluminescence reagents ECL Western Blotting Detection System (Amersham Piscatawya, NJ, USA), in accordance with the manufacturer’s recommendations, followed by autoradiography.