Image laboratory 5

Cells were lysed using RIPA buffer (Pierce Inc, Thermo Scientific, USA) containing 1 × Halt Protease Inhibitor Cocktail (Pierce Inc., Thermo Scientific). Samples containing 30 µg of total protein were electrophoretically separated and blotted using the Bio-Rad V3 Western Workflow system according to the manufacturer’s recommendation. Immunoblotting was performed using anti-LIN28B rabbit monoclonal antibody (Ab191881, 1:2000; Abcam, Cambridge, MA). The primary antibody was incubated overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit was used as the secondary antibody, whereas HRP-conjugated anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (1:1000; Invitrogen) was used as a loading control. Chemiluminescent detection was performed using WesternSure Chemiluminescent Substrate (LI-COR, Lincoln, NE, USA). Band intensities were quantified using the band quantification tool in Image Laboratory 5.0 software (Bio-Rad Laboratories).

DNA was isolated using RNA/DNA/Protein Purification plus Kit (Norgen Biotek Corp., ON, Canada) according to manufacturer’s instructions. Briefly, DNA samples was denatured with 0.1 M NaOH at 99 °C for 5 min, neutralized with ammonium acetate, NH4OAc (6 M) as previously described [35 (link)]. Different concentration of DNA (20 to 100 ng) in 5 µL blotted onto nitrocellulose membranes (GE Healthcare, Life Science, Germany). The DNA spots was dried at 60 °C for 10 min and further UV-cross linked (60 s, 1200 J/cm2), followed by blocked with 5% non-fat dry milk in tris-buffer saline (TBS) at room temperature for one hour. Subsequently, the level of DNA methylation was analyzed using anti- 5-methylcytosine (5-mC) mouse monoclonal antibody at 1:1000 dilution (Catalogue No: 39649, Active Motif, CA, USA) at overnight at 4 °C incubation. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse was used as the secondary antibody at 1:2000 dilution. Chemiluminescent detection was performed using WesternSure Chemiluminescent Substrate (LI-COR, Lincoln, NE, USA). Intensities were quantified using the quantification tool in Image Laboratory 5.0 software (Bio-Rad Laboratories).