Ripa protein extraction buffer

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/).

RIPA protein extraction buffer (Sigma‐Aldrich, St. Louis, MO, USA), supplemented with a protease inhibitor cocktail (Sigma‐Aldrich, St. Louis, MO, USA), was used to extract total protein, determined subsequently by the BCA Protein Assay Kit (Pierce Thermo Scientific, USA). The electrophoresis was performed utilizing 10 µg of protein in denaturing polyacrylamide 4–20% NuPage gel (Invitrogen, Carlsbad, CA, USA) for 60 minutes (140 V and 110 mA). After that, the specimens were transferred into a nitrocellulose membrane with the eBlot Protein Transfer System (Genscript, Piscataway, NJ, USA).
The membrane was incubated for one hour at room temperature with 5% nonfat milk dissolved in TBST. Subsequently, the membrane was incubated with primary antibodies (mouse) for 12 hours at 4°C and then with secondary anti‐mouse IgG secondary antibodies (goat), conjugated with alkaline phosphatase for 90 minutes at room temperature. All the antibodies were purchased from Santa Cruz Biotechnology, Dallas, USA. The bands on the membrane were developed using a BCIP/NBT alkaline phosphatase substrate (Merck Millipore, Darmstadt, Germany) and after that analysed with Image J 1.49 software (Wayne Rasband, National Institutes of Health, Bethesda, MD).

Protein expression levels were analyzed as previously described^{18 (link),51 (link),53 (link)}. Briefly, total proteins were extracted from hPSCs in ice-cold RIPA protein extraction buffer (SIGMA) supplemented with protease inhibitors. 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. 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 revealed by enhanced chemiluminescence detection (SUPERSIGNAL WEST FEMTO SYSTEM, THERMO SCIENTIFIC). For information about antibodies used please see Supplementary methods (Table S2). Densitometric analysis of protein levels were performed with IMAGEJ 1.34S SOFTWARE (WAYNE RASBAND, NATIONAL INSTITUTES OF HEALTH, https://imagej.nih.gov/ij/).