Protein Extraction and Western Blotting

For protein extraction, cells were collected together with medium and centrifuged at 1600 rpm at 4°C for 5 min. Pellets were washed twice with ice-cold PBS and then re-suspended in 200μl lysis buffer [9 (link), 10 (link)]. Protein concentration was assessed using Protein Assay Reagent (Thermo Scientific, USA). Western blotting was done by electrophoresing 10μg proteins on SDS–PAGE and subsequently transferring electrophoretically onto nitrocellulose membranes (Optitran, GE Healthcare Life Sciences, UK). Blocking was done in 5% non-fat dry milk in TBST for 2-4h at room temperature and then probed with appropriate primary and secondary antibodies. For details, please see Suppl. material.