Quantification of Placental Proteins

The total protein concentration in placental homogenates was determined using the Bradford assay (Bio-Rad). The placental homogenate (10 μg) was separated on 4%–20% precast linear gradient gels (Invitrogen). Membranes were incubated overnight at 4°C with primary antibody diluted in 1% non-fat milk (wt/vol) in TBST and detected using an appropriate peroxidase-conjugated secondary antibody. Products were visualized by ECL chemiluminescence (Millipore). Band intensities were measured using the G-box system (Syngene). Anti-β actin was purchased from Sigma-Aldrich, St. Louis, Mo. Target band densities were normalized using beta-actin to account for any variation in loading and transfer. Additional protein expression analysis was performed using automated capillary-based immunoassay (ProteinSimple, San Jose, CA, catalog #SM-W004-1, #PS-ST01, and #PN-009–050), as described previously (Castillo-Castrejon et al., 2021 (link)). Briefly, Jess plates were run according to the manufacturer’s instructions, with minor modification (200 V, 55 min separation time) with 0.1 mg/mL total protein concentration.