Anti cyclophilin b

TSA was purchased from Enzo Life Sciences. Carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC) and 7-amino-4-trifluoromethyl coumarin (AFC) were from Enzyme Systems Products (Livermore, CA). Unless indicated, all other reagents including cisplatin and chloroquine were purchased from Sigma (St. Louis, MO). The following primary antibodies were used: anti-LC3B from Novus Biologicals (Littleton, CO); anti-ATG7, anti-β-actin and anti-cyclophilin B from Abcam; and anti-cleaved caspase3, anti-AMPK, anti-phospho-AMPK (Thr172), anti-P70S6K, anti-phospho-P70S6K (T389) from Cell Signaling Technology (Danvers, MA). All secondary antibodies for immunoblot analysis were from Thermo Scientific (Rockford, IL).

Western blot analysis of NF-κB and MAPK was performed in the fetal membrane samples. Tissues were lysed in RIPA buffer (J63306: Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor (165-26021: Fuji film, Osaka, Japan), and 1000 μg of protein extracts were subjected to electrophoresis using 4–12% SDS-PAGE gels (NW04127: Thermo Fisher Scientific, Waltham, MA, USA). Proteins were transferred to membranes using an iBlot2 Dry Blotting System (IB21001: Thermo Fisher Scientific, Waltham, MA, USA). The membranes were incubated overnight with the following primary antibodies: anti-phosphorylated-NF-κB p65 (1:1000; #3033: Cell Signaling Technology (CST), Danvers, MA, USA), anti-NF-κB p65 (1:3000; #8242: CST), anti-phosphorylated-JNK (1:1000; #4668: CST), anti-JNK (1:1000; #9252: CST) anti-phosphorylated-p38 MAPK (1:3000; #4511: CST), anti-p38 MAPK (1:3000; #8690: CST), and anti-cyclophilin B (1:20,000; ab178397: Abcam, Cambridge, UK). Cyclophilin B was used as a loading control. Protein bands were densitometrically analyzed using Image Saver 5 for the Ez-Capture series (ATTO Corporation, Tokyo, Japan). Image J version 1.51 was used to calculate the relative densities of the bands (National Institutes of Health, Bethesda, MD, USA).