Peroxidase conjugated anti rabbit secondary antibody

Abcam (Cambridge, UK) provided the anti-glyoxalase 1 (GLO1) (cat. Ab171121; dilution: 1:1,000) and anti-glyoxalase 2 (GLO2) (cat. Ab154108; dil. 1:250) antibodies. Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) provided the anti-RAGE (cat. sc-365154; dilution: 1:250) antibody. The anti-α-tubulin antibody (cat. T5168; dilution 1:8000) and the peroxidase-conjugated anti-mouse secondary antibody (cat. A9044; dil. 1:10,000) were purchased from Sigma–Aldrich (Milano, Italy). Vector Laboratories (Peterborough, UK) provided the peroxidase-conjugated anti-rabbit secondary antibody (cat. PI1000; dil. 1:1000).

Tissue lysates obtained from PE and GA-matched control placental samples containing decidua basalis (n = 4/each group) as well as cell lysates obtained from control- and ISG15-siRNA transfected HTR8/SVneo cells subjected to SDS-PAGE (Bio-Rad, Hercules, CA, United States), and transferred onto a nitrocellulose membrane. Membranes were first blocked with 5% non-fat dry milk in TBS with 0.1% Tween 20 (TBS-T) for 1 h at room temperature, then incubated with mouse-anti-ISG15 (1:500; Santa Cruz) overnight at 4°C. Several washing steps of membranes followed incubation with peroxidase-conjugated anti-mouse secondary antibody (1:5000; Vector Labs) for 1 h at room temperature. An enhanced chemiluminescence kit (Thermo Fisher Scientific) was used to detect immunoblot bands. After stripping, the membrane was re-probed with HLA-G (1:1000: Cell signaling) overnight at 4°C followed by 1 h room temperature peroxidase-conjugated anti-rabbit secondary antibody (1:5000; Vector Labs) incubation. For endogenous loading control, membranes were then re-probed with HRP-conjugated rabbit-anti-β-actin (1:1000; Cell Signaling) antibody for 30 min at room temperature. Densitometric quantification was performed via Image J software (ImageJ 1.52a. National Institutes of Health, Bethesda, MD).