Anti g9a

Human glioblastoma cells or tumor tissues were harvested and washed with ice-cold PBS. Then, samples were suspended in SDS sample buffer, boiled for 10 min, and centrifuged at 12,000 rpm for 15 min. Thirty micrograms of protein samples were separated using 8–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore Corporation). The PVDF membrane was blocked with 5% milk for 1 h and then incubated with a primary antibody at 4°C overnight. Subsequently, the PVDF membrane was probed with the secondary antibody and visualized by enhanced chemiluminescence (ECL) (Beyotime Institute of Biotechnology, Haimen, China). The primary antibodies were as follows: anti-G9a (1:500, Santa Cruz), anti-a-tubulin (1:2000, Sigma), anti-CDK1 (1:1000, Abcam Cambridge, MA, United States), anti-CDK2 (1:500, Santa Cruz), anti-Cyclin A2 (1:2000, Abcam), anti-Cyclin B1 (1:3000, Abcam), anti-MMP1 (1:1000, Abcam), anti-N-cadherin (1:5000, Abcam), anti-LC3B (1:1000, Cell Signaling Technologies, Danvers, MA, United States), anti-p62 (1:1000, Abcam), anti-c-Myc (1:1000, Abcam), and anti-H3K9me2 (1:2000, Abcam). Horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000), goat anti-rabbit IgG (1:20,000), and rabbit anti-goat IgG (1:10000) (KPL, Gaithersburg, Maryland, United States) were used as secondary antibodies.

Western blot analysis was performed with the primary antibodies including anti-G9a (1∶500, Santa Cruz), anti-α-tubulin (1∶2000, Sigma-Aldrich), anti-H3K9me2 (1∶500, Abcam, Cambridge, MA, United States), anti-CDK2 (1∶500, Santa Cruz), anti-CDK4 (1∶500, Santa Cruz), anti-CDK6 (1∶500, Santa Cruz), anti-CyclinD1 (1∶500, Santa Cruz), anti-CyclinE (1∶500, Abcam), anti-LC3B (1∶500, Cell Signaling Technologies), and the autophagy antibody sampler kit (1∶500, Cell Signaling Technologies). Horseradish peroxidase-conjugated goat anti-mouse (1∶20000), goat anti-rabbit (1∶20000), and rabbit anti-goat (1∶10000) immunoglobulin G (IgG; KPL, Gaithersburg, Maryland, United States) were used as secondary antibodies.