Antiactin

Cell lysates were prepared from 4 hpf and 24 hpf UIC, wnt10a Tb‐ and SbMO‐injected embryos, and wnt10a RNA‐injected embryos, and dechorionated with pronase. After de‐yolking manually on an agarose plate, the embryos were collected and mixed with SDS sample buffer and boiled for 10 min. Proteins were resolved by SDS‐PAGE electrophoresis (4–12% gradient, NuPAGE Novex Bis‐Tris Gel 1.0 mm) (Life Technologies, Foster City, CA) and then transferred onto PVDF membrane (Invitrogen iBlot Dry, Carlsbad, CA). The membrane was incubated with primary antibodies anti‐wnt10a (cat. ARP41277_P050, LI‐COR Biosciences, Lincoln, NE) and antiactin (cat. Ab8227, Abcam, Cambridge, UK) at room temperature for 2 h. Following washing three times with 1xTBST for 15 min each, the membrane was incubated with IRDye^® 800 CW antirabbit IgG secondary antibody (cat. 926‐32211, LI‐COR) for 1 h at room temperature. Protein bands were visualized using LI‐COR Odyssey Scanning Imager (LI‐COR).

Placental STBMs, isolated from CTRL (n = 4) or PE patients (n = 4), were lysed. After boiling, 20 µg of STBMs, diluted in Laemmli buffer, were subjected to a 10% v/v SDS-PAGE under reducing conditions. Proteins were transferred to a nitrocellulose membrane using the wet system of Mini Blot Module (Thermo Fisher Scientific), at a constant voltage for 60 min. An hour of blocking step with 5% skimmed milk in Tris-Buffered Saline + Tween 20 (TBST; 10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) was followed by an overnight incubation at 4°C with primary antibodies: anti- FH (1:1000; #PA5-83957, Invitrogen) and anti-actin (1:1000; sc-8432, Santa Cruz). The following day, the membrane was incubated with anti-rabbit LI-COR IRDye 800CW and anti-mouse LI-COR IRDye 680CW (1:10,000; LI-COR Biosciences, Lincoln, NE, USA), for 1 h at room temperature. The fluorescence intensity was acquired in the Odyssey® CLx near-infrared scanner (LI‐COR Biosciences, Lincoln, NE, USA) and normalized for anti-actin. Image acquisition, processing and data analysis were performed via Image Studio Ver 5.2 (LI-COR Biosciences).