Ecl detection kit

OCI‐Ly1 and OCI‐Ly19 cells were lysed at 4 °C in lysis buffer and were electrophoresed in 10% SDS/PAGE and then transferred to an NC membrane (Millipore, Billerica, MA, USA). The membranes were blocked for 1.5 h with 5% nonfat milk in TBS‐T and then probed with primary antibodies (1 : 1000 in 5% bovine serum albumin in TBS‐T) overnight at 4 °C according to the manufacturers, followed by secondary HRP‐conjugated antibody (1 : 20000; CST, Beverly, MA, USA) and visualised with an ECL detection kit (Biological Industries, Beit HaEmek, Israel). The primary antibody against EDN1 was purchased from Abcam (Cambridge, MA, USA). VEGFR‐2, p‐VEGFR‐2, ERK, p‐ERK, P38, p‐P38, MEK, p‐MEK, Caspase 3, PARP, BAX, BIM, BCL‐2, MCL‐1 and β‐actin were purchased from Cell Signaling Technology (Danvers, MA, USA).

Placental tissue processing was performed as described by Sammar et al. [31 (link)]. Briefly, placental tissue was homogenized and solubilized in RIPA lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 20 mM Tris/HCl (pH 8.0), 150 mM NaCl) containing a complete set of protease inhibitors (Hoffman la Roche, Switzerland) for 30 min at 2–8 °C. Insoluble material was removed by centrifugation and protein concentration was determined by BCA reagent (Pierce, Rockford, IL, USA). Placental lysate samples were aliquoted and stored at −70 °C until use.
For Western blot analysis, 50 µg of total protein lysates was separated on 12.5% SDS-PAGE and electro-transferred to a nitrocellulose membrane. After blocking free binding sites with 5% non-fat milk in 20 mM Tris/HCl buffer at pH 8.0 supplemented with 150 mM NaCl, membranes were probed with the anti-CD24 mAb SWA11 and anti-β-actin antibodies (0.1 µg/mL) overnight at 4 °C. Bound immune complexes were detected by horseradish peroxidase-conjugated rabbit anti-mouse IgG and developed by ECL detection kit (Biological Industries, Beit Haemek, Israel). β-Actin was used as reference housekeeping protein for equal protein loading and densitometry analysis. Signals were developed by chemiluminescence and were captured by an imager (Bio-Rad, Hercules, CA, USA).