Adi 900 173

Corticosterone was extracted from tail venesection samples and measured by commercial ELISA kit (ADI-900-097; Enzo Life Sciences, UK). For copeptin and aldosterone, mice were euthanized by decapitation (between 7am and 8am), trunk blood was collected on ice, and separated by centrifugation and stored at −20⁰C. After a single thaw of all samples, copeptin (CEA365Mu; Cloud-Clone Corp., USA) and aldosterone (ADI-900-173; Enzo Life Sciences, UK) were measured by commercial ELISA.

Sera and placental samples were analysed using commercially available enzyme-linked immunosorbent assays (ELISA). Angiotensin II (ADI-900-204, Enzo Life Sciences, Inc., Farmingdale, NY, USA), Flt-1 (ab270206, abcam, Cambridge, UK) and aldosterone (ADI-900-173, Enzo Life Sciences, Inc., Farmingdale, NY, USA). ELISAs were conducted according to the manufacturer’s instructions. Briefly, serum samples were diluted (1:4) with the provided diluent and loaded onto the 96-well plates. Placental samples were prepared by homogenizing half of the placenta in lysis buffer (1:10 w/v) containing protease inhibitors. Homogenates were centrifuged at 13,000 revolutions per min for 20 min at 4 °C and supernatants collected. Total protein concentration was determined using a Pierce™ BCA Protein Assay Kit (23227, Thermo Scientific™, Waltham, MA, USA). Individual proteins were calculated relative to total protein content in homogenates. The concentrations were calculated using 4 parametric logistic regression (4PL) curve methods.