Protein Expression Analysis in Placenta

Total protein from both the placental samples and trophoblast cells was extracted using RIPA lysis buffer, and quantified using the Bradford method (Thermo Fisher Scientific, Waltham, MA, USA). A total of 20 μg protein of each sample was electroblotted onto PVDF membranes (Millipore, Burlington, MA) after sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation [12 (link)]. Protein signals were developed using an enhanced chemiluminescence kit (Bio-Rad, Irvine, CA) and quantified with a ChemiDoc system (Bio-Rad, Irvine, CA). The primary antibodies and dilutions are listed as following: rabbit polyclonal antibodies against adiponectin (1:500, Proteintech, Wuhan, China), fatty acid binding protein 4 (FABP4) (1:1000, Abcam, Cambridge, UK), peroxisome proliferator activated receptor gamma (PPARg) (1:1000, Cell Signaling Technology, Boston, MA, USA), acetyl-CoA carboxylase (ACC) (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho-ERK1/2 (1:1000, Cell Signaling), low density lipoprotein receptor (LDLR) (1:1000, Thermo Fisher), mouse monoclonal antibodies against sterol regulatory element-binding protein 2 (SREBP2) and sterol regulatory element-binding protein 1 (SREBP1) (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (1:5000, Sigma-Aldrich) and sortilin 1 (SORT1) (1:1000, BD Bioscience, Ann Arbor, MI, USA).