Mini protean tube cell instrument

Protein lysates were generated using the mammalian protein extraction reagent RIPA (Beyotime, Shanghai, China). The concentration of extracted total protein from each sample was calculated using the BCA Protein Assay Kit (Thermo Pierce, Rockford, IL, USA). The equivalent protein for each sample was loaded into a 10% sodium docedyl sulfate-polyacrylamide gel electrophoresis and fractionated, and the denatured proteins were subsequently transferred from gel to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) by a Mini-PROTEAN Tube Cell instrument (Bio-Rad, Hercules, CA, USA). The membranes were incubated with antibodies (IGF-1, ab9572, Abcam; glyceraldehyde 3-phosphate dehydrogenase, ab8245, Abcam) overnight at 4°C and then with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 1 h at room temperature. The enhanced chemiluminescence substrate (Beyotime) was used to visualize the band, and a picture was captured by an imaging system (UVP, Upland, CA, USA). Finally, the quantification analysis was performed by ImageJ 1.45 software (NIH Image).

Western blot analyses were carried out as described previously [17 (link)]. The total protein of the placenta samples was extracted using a Tissue Protein Extraction Kit (CWBIO, Jiangsu, China), and the concentration of the extracted total protein was determined by a bicinchoninic acid assay (CWBIO, Jiangsu, China). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transmembrane experiments were carried out with the Mini-PROTEAN Tube Cell instrument (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies, including anti-FATP4 (ProteinTech, Rosemont, IL, USA), anti-CD36 (ProteinTech, Rosemont, IL, USA), anti-FABP5 (ProteinTech, Rosemont, IL, USA), and anti-β-actin primary antibodies (Cell Signaling Technology, Danvers, MA, USA). Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz company, Hercules, CA, USA), and immunoreactive bands were visualized using the ECL kit (Beyotime, Shanghai, China) and documented with a chemiluminescence imaging system (UVP, Upland, CA, USA). The results of the densitometric analysis of bands were quantified by Image J 1.45 software (National Institutes of Health, Bethesda, MD, USA). To calculate the relative expression levels of the target proteins, all blots were standardized to β-actin.