Ab106368

RIPA lysis buffer (Takara Biotechnology, Dalian, China) was used to extract total proteins from cells. A total of 20 µg proteins were isolated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA). After blocked in the TBS buffer containing 5% skim milk (50 mmol/L NaCl, 10 mmol/L Tris, pH7.4), the membranes were washed with TBST for three times at 5 min each time and incubated with the primary antibodies at 4 overnight. Primary antibodies were DNMT1 (ab188453, 1:1000, Abcam, UK), OTUD4 (ab106368, 1:500, Abcam, UK) and GAPDH (ab181602, 1:10,000, Abcam, UK). Then, the membranes were washed with TBST as above procedures, and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h. Finally, immunoreactive proteins were treated with enhanced chemiluminescence reagent (Amersham, Little Chalfont, UK) and protein bands were observed using Amersham Imager 600 system (GE Healthcare Life Sciences, Shanghai, China).