Amersham ecltm prime

PBS-washed cell pellets were resuspended in 100 μL of radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Seoul, Korea) containing protease inhibitor cocktail solution (GenDEPOT, Seoul, Korea) and phosphatase inhibitor cocktail solution (GenDEPOT, Seoul, Korea) and incubated at 4 °C for 30 min. The total protein concentration was determined with the Bradford assay reagent (Bio-Rad, Seoul, Korea) at a wavelength of 595 nm (TECAN, Männedorf, Switzerland). Equal amounts of protein (10 μg) were electrophoresed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Seoul, Korea). The membrane was saturated with 5% bovine serum albumin (BSA) and incubated with 1:1000 diluted primary antibody including PPARγ, C/EBPα, FAS, SCD-1, FAT, and ACC (Cell Signaling Technology, Danvers, MA, USA) and β-actin (Santa Cruz Biotech, Seoul, Korea) at 4 °C for 14 h. Western blot signals were visualized using 1:5000 diluted horseradish peroxidase (HRP) conjugated secondary antibodies and developed with Amersham^TM ECL^TM prime (GE health care, UK), and then scanned using a C-Digit blot scanner (LI-COR Biosciences, Lincoln, NE, USA). Protein expression levels were quantified by Image J software (National Institutes of Health, Bethesda, MD, USA).

Aliquots of placenta homogenate (80 µg total protein) were mixed with loading buffer under reducing conditions [71 (link)], heated at 96 °C for 5 min; and separated by SDS-PAGE on 10% (SERT, OCT3), 12.5% (MAO) or 15% (IDO and TPH) polyacrylamide gels. Electrophoresis was performed at 120 V and proteins were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 20 mM Tris-HCl pH 7.6, 150 mM NaCl, and 0.1% Tween 20 (TBS-T) containing 5% BSA for 1 h at room temperature and washed with TBS-T buffer. Incubation with primary antibodies (listed in Supplementary Table S2) was performed overnight at 4 °C against SLC6A4, SLC22A3, MAO, IDO, and TPH. After washing with TBS-T buffer, the membranes were incubated with a specific secondary antibody (listed in Supplementary Table S2) for 1 h at room temperature. Membranes were developed using Amersham^TM ECL^TM Prime (GE Healthcare Life Science, Marlborough, MA, USA). The band intensity was visualized and quantified by densitometric analysis using the ChemiDoc^TM MP, Imaging system (Bio-Rad, Hercules, CA, USA). To ensure the equal loading of proteins, membranes were probed for β-actin and specific secondary antibodies (listed in Supplementary Table S2).