Bca assay kit

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.

Total cellular proteins were extracted using RIPA lysis buffer supplemented with a proteinase inhibitor cocktail, and the concentration of proteins in the centrifuged lysis buffer was determined using a commercially available BCA Assay Kit (Bio-Rad, Hercules, CA, USA). Total protein (20 μg) in the supernatant was mixed with 2× SDS loading buffer and then loaded onto a 10–12% precast Bis-Tris gel (Invitrogen). After electrophoresis, the separated proteins were transferred to PVDF (polyvinylidene difluoride) membranes (Millipore, Billerica, MA, USA) and blocked with 5% skim milk powder at room temperature for 60 min. Then the membranes were incubated with 1.0 μg/ml primary antibodies against LDHA, γ-H2AX, and β-actin (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C, respectively. Afterward, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000 dilution) for 60 min at room temperature. The target proteins were indicated using ECL Western blotting detection reagents (ECL New England Biolabs, Beverly, MA, USA). Quantification of the target proteins was performed using a Bio Image Intelligent Quantifier 1-D (Version 2.2.1; Nihon-BioImage Ltd., Tokyo, Japan) with β-actin used as an internal control.

Tissue samples were lysed with RIPA buffer (Dingguo, Inc., Beijing, China) for protein extraction. Protein concentration was determined by the BCA assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts (10 µg) of total protein was loaded onto SDS–PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes, which were prepared by formaldehyde activation for 5 minutes and being lysed in Tris-buffered saline for 10 minutes. Subsequently, the membranes were incubated in blocking buffer (5% non-fat milk) for 1 hour, then immunoblotted overnight at 4°C with primary antibodies against SP1 (Abcam) or GAPDH (Bioleaf, Shanghai, China). After washing the membranes three times for 15 minutes with TBST [10 mM Tris–HCl (pH 7.4), 0.5 M NaCl, Tween 20 (0.1% v/v)], the membranes were incubated for 90 minutes with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:2000; Invitrogen), followed by washing the membranes three times for 10 minutes with TBST. Immunoreactive bands were detected by the enhanced chemiluminescence system (Pierce, Rockford, IL, USA) and quantifications were estimated using Image Lab 4.1 software (Bio-Rad Laboratories, Hercules, CA, USA).