Bba16

Tissues were homogenized using TissueLyser II (Qiagen) according to manufacturer’ instructions. Proteins were isolated using RIPA buffer (Boston BioProdcuts, BP-115) with protease inhibitor and phosphatase inhibitors. Protein concentrations were determined using Pierce BCA assay (Thermo Scientific). Twenty μg protein were loaded per lane on a 4–20% Mini-PROTEAN TGX Gel (Bio-Rad, 456–1096). Separated proteins were transferred to PVDF membranes using the Transfer Turbo Blot system (Bio-Rad) and Trans-Blot Turbo RTA Transfer Kit (Bio-Rad, 170–4272). The membrane was blocked with 5% nonfat milk in TBST for 1 hr at room temperature. After blocking, the membrane was incubated overnight at 4°C with antibodies against ADORA1 (1:500, ab82477, abcam), ADORA2A (1:750, ab3461, abcam), ADORA2B (1:750, ab40002, abcam), ADORA3 (1:500, ab203298, abcam), VCAM-1 (1:1000, ab134047, abcam), ICAM-1 (1:3000, BBA3, R and D Systems), E-Selectin (1:1000, BBA16, R and D Systems), p-AKT (1:1000, 4060, Cell Signaling), T-AKT (1:1000, 2920, Cell Signaling), β-actin (1:4000, 4970, Cell Signaling). Quantification of protein bands were performed using a luminescent image analyzer (BioRad, Chemidoc).

Immunohistochemical analysis was performed as previously described (37 (link)), using antibodies to E-selectin (mouse monoclonal, BBA16; 5 μg/mL; R&D Systems), P-selectin (mouse monoclonal, BBA30; 10 μg/mL; R&D Systems) and L-selectin (rabbit polyclonal, GTX59778; 0.1 μg/mL; GeneTex Inc.). All slides were assessed by the same observer, blinded to pregnancy outcome. Quantification was performed as described previously (12 (link)), using the Positive Pixel Algorithm of Aperio ImageScope software; a visual check was also performed to establish localization of staining. The AT1R and AT2R protein expression analysis was performed as previously reported (14 (link)).