Total Protein Extraction and Immunoblotting

For total protein extraction, iLIN28B and CTRL cells were trypsinized (PAN Biotech, Aidenbach, Germany), washed with cold PBS once, and resuspended in cold lysis buffer (Biosource International, Camarillo, CA, USA) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Immunoblotting was performed with 20 µg of the proteins quantified with Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s recommendations. Proteins were loaded on 4–20% gradient gel and SDS-PAGE (Bio-Rad, Hercules, CA, USA) was performed as described elsewhere [36 (link)]. Primary antibodies used in the study were anti-LIN28B (Cell Signaling, Danvers, CA, USA; 4196S) and anti-Vinculin (Santa Cruz, Dallas, TX, USA; sc-73264). For protein visualization, secondary horseradish-peroxidase (HRP)–conjugated antibodies (Sigma-Aldrich) were used. Enhanced chemiluminescence (ECL) Western blotting detection reagents (ECL^TM Select, Merck Life Science S.r.l., Milan, Italy) were used for protein band acquisition with the iBright Imaging Systems (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA).