Supersignal west femto system

Cells were cultured and retrieved in RIPA reagent. The cell lysate was centrifuged for 15 min at 15 000g, and the proteins were collected. After protein quantification by BSA Assay Kit, WB was implemented. The antibodies for GAPDH (Sigma; dilution at 1:5000, Shanghai, China), active β‐catenin (Merck Millipore, 05665; dilution at 1:3000, Shanghai, China) and total β‐catenin (BD Biosciences, 610154; dilution at 1:3000, Shanghai, China) were utilized. The signal intensity was detected and quantified by SuperSignalWest Femto system (Thermo Scientific, Shanghai, China).

Protein expression levels were analyzed as previously described^{5 (link)}. Total proteins were extracted from hPSCs in ice-cold RIPA protein extraction buffer (Sigma) supplemented with protease inhibitors (Protease inhibitor cocktail set I, Calbiochem). Protein concentration was determined using Bicinchoninic Acid Protein Assay (Pierce). Equal amounts of protein were electrophoresed on a 12% SDS–polyacrylamide gel and transferred to PVDF membranes (Millipore). Blots were blocked 1 h at RT (room temperature) in TBS (20 mM Tris–HCl, pH 7.5, 500 mM NaCl) containing low-fat powdered milk (5%) and Tween 20 (0.1%). Incubations with primary antibodies were performed ON (overnight) at 4 °C in blocking buffer (3% skim milk, 0.1% Tween, in Tris-buffered saline). Membranes were then incubated with the corresponding counter-antibody and the proteins were revealed by enhanced chemiluminescence detection (SuperSignal West Femto System, Thermo Scientific). In most cases, full-length blots are not provided in Supplementary information as blots were cut before hybridization with antibodies to save on samples and reagents. For information about the antibodies used please see Supplementary methods (Table S2). Densitometric protein level analysis was performed with ImageJ 1.34 s software (https://imagej.nih.gov/ij/).

Syncytin‐2 expression plasmids were transfected into 293T and G355‐5 cells in the same manner as cell fusion assays. Cells were lysed in RIPA Lysis Buffer (#08714‐04; Nacalai Tesque) from transfection to G355‐5 cells at 7 h and from transfection to 293T cells at 20 h. Cellular suspensions were subjected to glycolysis treatment for 1 h using a PNGase F Kit (New England BioLabs). SDS/PAGE was performed using Mini‐PROTEAN TGX Precast Gels (#4561094; Bio‐Rad Laboratories, Inc., Hercules, CA, USA). Peptides from the gel were transferred to polyvinylidene difluoride membranes, and the monoclonal ANTI‐FLAG M2 antibody (#F3165; Sigma‐Aldrich) was used to detect Flag‐tagged syncytin‐2. Signals were detected using a Super Signal West Femto System (#34095; Thermo Fisher Scientific), and images were obtained using a LAS4000 Mini camera system (Fujifilm, Tokyo, Japan).