Enhanced chemiluminescence detection kit

For protein extraction, cells were lysed in cell lysis buffer containing 140 mM NaCl, 10 mM Tris-HCl, 1% Triton X-100, 1 mM ECTA and 1X protease inhibitor cocktail (Gibco; Thermo Fisher Scientific, Inc.). The protein concentration was determined using the BCA assay. A total of 20 µg protein was loaded per lane and separated on 12% SDS-PAGE gels. Proteins were subsequently transferred to polyvinylidene difluoride membranes, which were blocked with 5% non-fat dry milk for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-GAPDH (cat no. sc-25778; 1:2,000) anti-BMP6 (cat no. sc-7406; 1:100; both Santa Cruz Biotechnology, Inc.) and anti-VEGF (cat no. ab105219; 1:1,000; Abcam). They were subsequently incubated with an anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (cat no. sc-2004, 1:1,000, Santa Cruz Biotechnology, Inc.) at 4°C for overnight, and detected with an enhanced chemiluminescence detection kit (EMD Millipore, Billerica, MA, USA).

The placental tissues were homogenised on ice in radio-immune precipitation assay (RIPA) lysis buffer containing 50 mM Tris-HCl (pH 7.6), 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), phenylmethylsulphonyl fluoride (PMSF), 1 mg/L aprotinin and 1 mg/L leupeptin. The samples were centrifuged and the protein concentrations of the supernatants determined using the BCA assay kit (Bio-Rad). Equivalent quantities of protein (50 µg) from each group were separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE). The separated protein bands were transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA) and blocked for 60 min at 37℃ using Tris buffered saline with Tween-20 (TBST) buffer (20 mM Tris pH 7.6, 137 mM NaCl and 0.1% Tween 20) with 5% non-fat milk. The membranes were incubated overnight at 4℃ with specific primary antibodies (1:1,000) and then washed with TBST three times. Finally, the membranes were incubated at 37℃ for 60 min with HRP-labelled secondary antibodies (1:2,000). The positive bands were detected using an enhanced chemiluminescence detection kit (Merck Millipore, Burlington, MA, USA) and analysed using a ChemiDoc XRS imaging system (Bio-Rad). β-Actin was used as an internal control to normalise protein concentrations.