Hla g

Zoledronic acid, LPS (lipopolysaccharide), puromycin, and G418 were obtained from Sigma‐Aldrich. Antibodies against human HLA‐G (sc‐21799) and β‐actin (sc‐47778) were purchased from Santa Cruz Biotechnology. PD‐L1 antibody (17952‐1‐AP) and PD‐L1 (22C3) were purchased from Proteintech and Agilent Technologies, respectively. Antibodies specific for CD3ε (NB600‐1441), PD‐L1 (FAB1561R, FAB1561G), CD4 (FAB3791R), and CD8 (NBP2‐34590AF488) were purchased from Novus Biologicals. HLA‐G (PE: #335906, Alexa Fluor 488: #335918), PD‐1 (#367412), CD3 (#317318), CD8 (#344704), CD56 (#304611), CD66b (#305104), and Vδ2 (#331418) were purchased from BioLegend. Anti‐Tyk2 (phospho‐Tyr1054/1055) (orb505746) antibody was obtained from Biorbyt. Antibodies specific for phospho‐ZAP70/Syk (Tyr319, Tyr352) (#MA5‐36963) and HLA‐G (MEM‐G/2: #MA1‐19394, 87G: MA1‐10356) were purchased from Thermo Fisher Scientific. Antibodies for CD14 (#12‐0149‐42), TCRγδ (#12‐9959‐42), TCRαβ (#11‐9955‐42), and NKG2D (#12‐5878‐42) were purchased from eBioscience. Vγ9 (#555732), hMito (ab92824), and VHH (iFluor 647: A02019, HRP: A2016) were purchased from BD Pharmingen, Abcam, and GenScript, respectively. Phospho‐Stat2 (Tyr690) (#77366) and HLA‐G (#79769) were obtained from Cell Signaling Technology. Recombinant human interleukin 2 (IL‐2) was obtained from Thermo Fisher Scientific.

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).