Hrp conjugated anti mouse igg or anti rabbit igg antibodies

DNMT3A KO and WT HEK293 cells of 1 ×10 6 were washed with PBS, lysed using 100 μL RAPA lysis buffer containing protease inhibitors cocktail (Roche; Penzberg, Germany), and separated by a 10% SDS-PAGE. After transferring onto a 0.45 μm PVDF membranes, immunoblotting was performed. For detection of DNMT3A deficiency, primary mouse monoclonal antibody against GAPDH (Sangon; Shanghai, China) and polyclonal rabbit-anti-human DNMT3A (Sangon) were used at 1:1000 dilution. For detection of MAPK and PI3K-Akt pathways, primary monoclonal antibodies against human Erk (137F5; Cell Signaling Technology; Danvers, MA, USA), phosphor-Erk (197G2; Cell Signaling Technology), JNK (D-2; Santa Cruz; Dallas, TX, USA), phosphor-JNK (G9; Cell Signaling Technology), Akt (11E7; Cell Signaling Technology), and phosphor-Akt (244F9; Cell Signaling Technology) were used. HRP-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (Jackson ImmunoResearch; PA, USA) were used for secondary antibodies. Signals were detected with enhanced chemiluminescence (Millipore; MA, USA) and visualized with a gel imaging system (Tanon; Shanghai, China).

DNMT3A KO and WT cells were cultured and seeded at 3×10 4 cells/well into 12-well plate. Cells were counted every 24 h for consecutive 6 days. And cell proliferation curves were compared between the two cell lines.
Western blot analysis DNMT3A KO and WT HEK293 cells of 1 ×10 6 were washed with PBS, lysed using 100 μL RAPA lysis buffer containing protease inhibitors cocktail (Roche; Penzberg, Germany), and separated by a 10% SDS-PAGE. After transferring onto a 0.45 μm PVDF membranes, immunoblotting was performed. For detection of DNMT3A de ciency, primary mouse monoclonal antibody against GAPDH (Sangon; Shanghai, China) and polyclonal rabbit-anti-human DNMT3A (Sangon) were used at 1:1000 dilution. For detection of MAPK and PI3K-Akt pathways, primary monoclonal antibodies against human Erk (137F5; Cell Signaling Technology; Danvers, MA, USA), phosphor-Erk (197G2; Cell Signaling Technology), JNK (D-2; Santa Cruz; Dallas, TX, USA), phosphor-JNK (G9; Cell Signaling Technology), Akt (11E7; Cell Signaling Technology), and phosphor-Akt (244F9; Cell Signaling Technology) were used. HRP-conjugated anti-mouse IgG or antirabbit IgG antibodies (Jackson ImmunoResearch; PA, USA) were used for secondary antibodies. Signals were detected with enhanced chemiluminescence (Millipore; MA, USA) and visualized with a gel imaging system (Tanon; Shanghai, China).