Western blot
The tissue and cells were lysed in ice-cold lysis buffer (KeyGEN
Biotech, Nanjing, China) followed by centrifugation at 4°C for 15 min.
Cell lysates were separated by 8% SDS‒PAGE and then transferred to
polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA).
After blocking with 5% nonfat milk, the membranes were incubated
overnight at 4°C with specific primary antibodies against the following
proteins: TP53BP2 (1:1000, Abcam, Cambridge, USA), LC3B (1:1000, Abcam,
Cambridge, USA), p62 (1:1000, Abcam, Cambridge, USA), Beclin-1 (1:1000,
Abcam, Cambridge, USA), Bcl-2 (1:1000, Abcam, Cambridge, USA), E2F1
(1:1000, Abcam, Cambridge, USA), DNMT1 (1:1000, Abcam, Cambridge, USA),
and G9a (1:1000, Abcam, Cambridge, USA). After three washes, the
membranes were incubated with horseradish peroxidase (HRP)-conjugated
secondary antibodies for 1 h, after which protein expression was
detected via a chemiluminescence kit (KeyGEN, Nanjing, China). The
optical density of each band was analyzed via densitometry and
normalized to that of a β-actin loading control.