Supersignal west pico detection reagent

Total protein samples (100μg per lane), isolated by sonication, were resolved by standard NuPAGE SDS-PAGE electrophoresis with MOPS running buffer (Life Technologies) and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with 1:10 Western Blocking Reagent (Roche) in TBS buffer with 0.1% of Tween-20 (TBST). The following day the membrane was incubated for 1h at RT with primary antibody solution in 1:20 Western Blocking Reagent diluted in TBST: rabbit polyclonal anti Lin28a (A177) (1:1000, Cell Signalling Technology), rabbit polyclonal anti Msi1 (N3C3) (1:1000, GeneTex), rabbit monoclonal anti hnRNP A1 (1:1000, D21H11) (Cell Signalling Technology), Lin28b (1:1000, Cell Signalling Technology), Tuj1 (1:20,000, GeneTex), GFAP (1:1000, SIGMA), DHX9 (1:1000, Protein-Tech), mouse-monoclonal anti–β-tubulin (1:10,000, Sigma). After washing in TBST, the blots were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase and detected with SuperSignal West Pico detection reagent (Thermo Scientific). The membranes were stripped using ReBlot Plus Strong Antibody Stripping Solution (Chemicon) equilibrated in water, blocked in 1:10 western blocking solution in TBST and re-probed as described above. Full scans of the western blots presented in the manuscript are shown in the Supplementary Fig 10.

After culture, cells were washed and lysed in complete Lysis-M supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany) for 10 min on ice. The lysates were centrifuged at 10,000Xg to remove cell debris. Alternatively, supernatants of treated cells were precipitated with 10% tricloroacetic acid (TCA) for 30 min on ice, centrifuged at 11,000Xg for 15 min and washed in cold acetone overnight at −20°C. Samples were centrifuged again at 11,000Xg for 15 min and resuspended in sample buffer (25%Tris pH 6.8, 10% SDS, 0.5% bromophenol blue, 10% glycerol) heated at 96°C for 10 min and quantified by the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). Aliquots corresponding to 20–25µl of protein were resolved in NuPAGE Novex 4–12% Bis-Tris Gels pre-cast polyacrylamide gels (Life Technologies) under reducing conditions and transferred to PVDF membranes (Life Technologies). Membranes were incubated with 5% non-fat dry milk (Santa Cruz) in TBS-T and then incubated with primary Abs followed by horseradish peroxidase (HRP)-conjugated secondary Abs. Proteins were visualized after exposure to Kodak Biomax (Sigma) using the enhanced chemiluminescense Super Signal West Pico detection reagent (Thermo Scientific, Rockford, IL). Immunoblotting for β-tubulin (mouse anti-human ascites, clone TUB 2.1, Sigma) was used as loading control.