Non reducing laemmli sample buffer

hiPSC-CMs were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (all from Sigma-Aldrich). Equal amounts of samples were mixed with non-reducing Laemmli sample buffer (BioRad, Hercules, CA, USA) and denatured at 96 °C for 5 min. Proteins were then separated on 10–12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Membranes were blocked with 5% non-fat dry milk powder in Tris-buffered saline. Primary antibodies used for western blotting were as follows: anti-tubulin (1/1000; T5168, Sigma-Aldrich), anti-caspase-3 (1/500; #9662, Cell Signaling Technology, Danvers, MA, USA), anti-cleaved caspase-3 (1/200; #9661, Cell Signaling Technology), anti-Bax (1/500; #5023, Cell Signaling Technology), anti-Bcl-2 (1/200; #4223, Cell Signaling Technology) and anti-phospho-Histone γ-H2AX (1/1000; 05-636-200, Merck Millipore). Secondary antibodies were anti-IgG rabbit (1/5000; P0448 Dako, Santa Clara, CA, USA) and anti-IgG mouse (1/5000; A9044, Sigma-Aldrich). Detection was carried out using peroxidase-conjugated antibodies and SuperSignal^TM West Femto (ThermoFisher Scientific). Reactions were visualized using an Amershan Imager 600 (GE Healthcare, Chicago, IL, USA) and quantified with the ImageJ software (ver. 1.53t, NIH, Bethesda, MD, USA).

Cells were lysed in RIPA buffer (1% NP40, 0,5% deoxycholate, 0,1% sodium dodecyl sulfate in Tris-buffered saline (TBS)) (Sigma-Aldrich) supplemented with protease (Complete, Sigma-Aldrich) and phosphatase (PhosSTOP, Sigma-Aldrich) inhibitors. Equal amounts of samples were mixed with non-reducing Laemmli sample buffer (BioRad, Hercules, CA, USA) and denatured at 96 °C for 5 min. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat dry milk powder in TBS. Primary antibodies used for western blotting were: anti-Smad2 (1/500, Abcam, Cambridge, UK, ab228765), anti-YAP (1/1000, Cell Signaling Technology, Danvers, MA, USA, D8H1X), anti-phospho-YAP (1/1000, Cell Signaling Technology, D9W2I) and anti-GAPDH (1/1000, Cell Signaling Technology, D16H11). Secondary antibody was anti-IgG rabbit (1/5000, Dako, Santa Clara, CA, USA, P0448). Detection was carried out using peroxidase-conjugated antibodies and SuperSignal^TM West Femto (Thermo Fisher Scientific). Reactions were visualized using an Amershan Imager 600 (GE Healthcare, Chicago, IL, USA) and quantified with ImageJ software (NIH, Bethesda, MD, USA).

EVs or cells were lysed in 100 μL of RIPA buffer containing protease (Complete, Sigma-Aldrich) and phosphatase (PhosSTOP, Sigma-Aldrich) inhibitors. Equal amounts of protein samples were suspended in non-reducing Laemmli sample buffer (BioRad) and denatured at 100°C for 5 min. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Membranes were blocked with TBS containing 5% (w/v) nonfat dry milk powder with 0.1% Tween-20. Antibodies used were anti-CD9 (Ab 92726, Abcam), anti-TGS 101 (Santa Cruz. Sc-7964), anti-Alix (Santa Cruz. Sc-53538) and HIF-1α (610958, BD biosciences). Detection was carried out using peroxidase-conjugated secondary antibodies with the ECL Plus Reagent (Amersham, GE Healthcare, Munich, Germany). Proteins were visualized using an Amershan Imager 600 (GE Healthcare) and quantified with ImageJ software (NIH).