Bis tris gradient gels

For SDS-PAGE analysis, precast 4–20% Bis-Tris gradient gels (Genscript, Picastaway, USA) in a Tris-MOPS running buffer was used. The molecular weight marker was Pierce™ Unstained MW marker (Thermo Scientific). Maximum volume of 20 µL was loaded in the wells on the SDS-PAGE gel. Solubilized extracts were mixed 14:5:1 with a 4xSDS sample buffer (50 mM Tris (VWR, Leuven, Belgium) pH 6.8, 2% SDS, 10% glycerol (VWR), 0.02% bromophenol blue (Sigma-Aldrich), 12.4 mM ethylenediaminetetraAcetic acid (EDTA, Carl Roth, Karlsruhe, Germany)) and 1 M dithiothreitol (DTT, Thermo Scientific), corresponding to a final DTT concentration of 50 mM. The samples incubated at 95 °C for 5 min to denature proteins. Running time for the gel was 45 min at 140 V. To visualize proteins, the gel was stained with Coomassie Brilliant Blue G250 (29 mM Coomassie Brilliant Blue G-250 (Sigma-Aldrich), 45% Ethanol (VWR), 10% Acetic acid (Fisher Scientific, Loughborough, UK)). Destaining of the gel was performed overnight with destain solution (8% Ethanol, 5% Acetic acid). Imaging of the gel was done using a ChemDoc MP Imaging System (Bio-Rad, Hercules, USA).

All iPSC pellets were lysed in lysis buffer containing 0.5% CHAPS, 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10% glycerol, 5 mM DTT, and 1 mM PMSF. Total lysates were incubated at 4 °C for 1 h on a rotator, and insoluble material was removed by centrifugation at 21,130 × g at 4 °C for 10 min. Protein concentration was measured using the protein assay dye (Bio-Rad, Cat. No. 5000006). Twenty micrograms of protein were resolved on 4–20% Bis-Tris gradient gels (GenScript, Cat. No: M00657) at 180 V for 40 min and then transferred to polyvinylidene fluoride (PVDF) membranes (BIO-RAD Immun-Blot®, Cat. #1620177) at 100 V for 1 h. Five percent skim milk in washing buffer was used as a blocking reagent. The prestained protein ladder (Omics Bio, Cat: 02101-250) was used as a marker; ɑ-tubulin (1:5000, ABclonal, Cat. No: AC012) was used as a loading control. Cytiva software was used. The antibodies used in this study are listed in Supplementary Table 7. All blots derive from the same experiment and were processed in parallel.