Lin28b

Western blots were performed as previously described [21] (link), using the following antibodies detecting: Akt (cat.# 4691, Cell signalling, dilution 1:1000), Dnmt1 (cat.# ab188453, Abcam; dilution 1:500), Dnmt3a (cat.# SC-20703, Santa Cruz; dilution 1:1000), Dnmt3b (cat.# PA1–884, Thermo Fisher; dilution 1:1000), Stat3 (cat.# SC-8019, Santa Cruz; dilution 1:500), P-Stat3 (cat.# 9145, Cell signalling, dilution 1:1000), Stat5 (cat.# 25656, Cell signalling, dilution 1:1000), P-Stat5 (cat.# 9359, Cell signalling, dilution 1:1000), mouse c-Met (cat.# SC-8057; Santa Cruz; dilution 1:500), c-Myc (cat.# SC-40, Santa Cruz; dilution 1:1000), Hsc-70 (cat.# SC-7298, Santa Cruz; dilution 1:10000), Lin28b (cat.# SC-293120, Santa Cruz; dilution 1:500), h-Ras (cat.# SC-520, Santa Cruz, dilution 1:1000) and β-actin (cat.# SC-130657, Santa Cruz; dilution 1:1000).

Total protein extracts were obtained from B16-F1 cells. After centrifugation at 14 000 g, proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (20 μg per lane) in a 10% polyacrylamide gel, transferred to polyvinylidene difluoride membranes and probed with primary antibodies specific for IL-6 (1:200; R&D Systems, Minneapolis, MN, USA), lin28B (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-Stat3, Stat3, p-p65, p65 or glyceraldehyde 3-phosphate dehydrogenase (1:2000; Cell Signaling, Danvers, MA, USA). After incubation with the appropriate horseradish peroxidase-conjugated antibody, the proteins were visualized using the enhanced chemiluminescence western blot analysis system (Amersham Biosciences, Piscataway, NJ, USA).