Mouse anti dnmt1

Total cellular proteins were extracted with 100 μL of radioimmunoprecipitation assay buffer containing protease inhibitor cocktail. After electrophoresis, the proteins were electrotransferred onto a nitrocellulose membrane that was blocked with 5% (w/v) nonfat dry milk for 1 h at 37 °C. The membranes were incubated with primary mouse anti‐Dnmt1 (dilution 1 : 2000), rabbit anti‐Dnmt3a (1 : 500), rabbit anti‐Dnmt3b (1 : 500) or mouse anti‐GAPDH (1 : 700, all purchased from Thermo Fisher Scientific) overnight at 4 °C. After washing, the membranes were incubated with anti‐mouse or anti‐rabbit horseradish peroxidase (HRP)‐conjugated secondary antibodies (Dako, Glostrup, Denmark) for 1 h at 20 °C. Proteins were detected using Clarity Western ECL substrate (Bio‐Rad, Hercules, CA, USA) and visualized using the Azure c600 imager (Azure Biosystems, Dublin, CA, USA).

For immunocytochemistry (ICC), cells were grown on eight‐well chamber slides and incubated with sarcosine (1 μm, 24 h). Then, cells were fixed with 4% paraformaldehyde for 15 min. After permeabilization with 0.3% Triton X‐100 in PBS for 3 min, the cells were blocked with 10% BSA in PBS and incubated with the indicated primary antibodies at 4 °C overnight. ICC was analysed with an EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). Rabbit anti‐GNMT (1 : 500) and rabbit anti‐SARDH (1 : 200) were from Abcam (Camridge, UK); mouse anti‐DMGDH (1 : 500) and mouse anti‐PIPOX (1 : 200) were from Santa Cruz Biotechnology (Dallas, TX, USA); and mouse anti‐Dnmt1 (1 : 1000) was from Thermo Fisher Scientific. ICC was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA), analysing the intensity of 80–120 cells per group with subsequent background subtraction (analysis without using primary antibodies).