Mouse anti isg15

The antibodies and reagents used in this study were commercially obtained from different sources, which were listed as follows: mouse anti-Influenza A NP, mouse anti-Influenza A NS1 and mouse anti-ISG15 (Santa Cruz); rabbit anti-F4/80 antibody and mouse anti-β-Tubulin (Abcam); rabbit anti-ISG15, rabbit anti-RIG-I, rabbit anti-MAVS, rabbit anti-MDA5, Alexa Fluor 488-cunjugated anti-Rabbit IgG (H+L), F(ab’)2 Fragment and anti-Mouse IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 647 Conjugate), anti-rabbit IgG (H+L) (DyLight 680 Conjugate), anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) and anti-mouse IgG (H+L) (DyLight 680 Conjugate) (CST); rabbit anti-RIG-I, mouse anti-Flag tag, mouse anti-AIF, rabbit anti-CD71, rabbit anti-Flotillin 1 and mouse anti-GAPDH (Proteintech); rabbit anti-ADAP (Millipore); mouse anti-ADAP (BD); mouse anti-HA tag (Sigma-Aldrich); IFN-β Protein (Sino Biology); LPS derived from Escherichia coli strain O111:B4 (Sigma-Aldrich); poly (I:C) (Selleck Chemicals). Mouse anti-viral protein (VP3) of IBDV polyclonal antibody was generated from the sera of mice by immunization with purified IBDV.

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).