Coomassie brilliant blue kit

Western blot was performed using placental tissue, which was homogenized and lysed using the RIPA buffer. Then, 50 μg of protein were loaded in each lane and SDS gels of 10–14% were used. Proteins were transferred to nitrocellulose membranes and blocked with 1% bovine serum albumin in phosphate-buffered saline (pH 7.4). All antibodies were acquired from Cell Signaling Technologies (Danvers, MA, USA) and used according to the recommendations of the vendors. Placental tissue sample was homogenized in 0.2% H3PO4 solution and then centrifuged for 10 min at 3500g at 4 °C to collect the supernatant before the enzyme-linked immunosorbent assay (ELISA). The ELISA kits were acquired from Abcam (Cambridge, MA, USA). The Coomassie brilliant blue kit (Jiancheng Institute of Biotechnology, Nanjing, China) was employed to detect proteinuria under the instructions from the manufacturer. Blood pressure was measured through a noninvasive tail-cuff method using the BP-2000 Blood Pressure Analysis System (Visitech Systems, Inc., Apex, NC, USA). Systolic blood pressure was assessed with five continuous values with variations < 6 mmHg averaged to define maternal systolic blood pressure.

The relative growth rates (RGRs) for the control and treatment groups were calculated from the following formula:³⁴ where W₀ refers to the initial FW and W_t is the FW at the end of the experiment. t is the cultivation time (15 days). SP was extracted at 0–4 °C in a phosphate buffer (50 mM, pH 5.5) by grinding 0.5 g FW with a mortar. The homogenates were then centrifuged (2000 rpm, 20 min, 4 °C), and the supernatants were used to determine the SP concentration.^{35 (link)} The analysis of SP concentration was performed with a Coomassie Brilliant Blue Kit according to the manufacturer's instructions (Jiancheng Biotech, Nanjing, China). The absorbance was read at 595 nm by UV-VIS spectrophotometry.