Ab2850

Total fraction proteins were separated using 10% SDS-PAGE with Tris-Gly buffer and transferred to 0.45 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) for 1 h at 250 mA on ice. The assessment of the quality of protein transfer, hybridization with primary and secondary antibodies, and protein development were performed as described previously. For primary hybridization, rabbit antibodies (Abcam, Cambridge, UK) to SUV39H1 (ab245380, 1:3000), SUV39H2 (ab229493, 1:3000), G9a (ab183889, 1:3000), DOT1L (ab64077, 1:500), EZH2 (ab228697, 1:7000), BRD2 (ab139690, 1:10,000), BRD3 (ab264294, 1:5000), BRD4 (ab128874, 1:1000), and DNMT3a (ab2850, 1:500); rabbit antibodies (Invitrogen, Carlsbad, CA, USA) SUV420H1 (PA5-40926, 1:3000) and SUV420H2 (PA5-109891, 1:3000); and rabbit antibodies DNMT1 (ABclonal, San Diego, CA, USA, A5495, 1:3000) were used. Goat antibodies (Abcam, ab97051, 1:10,000) were used for secondary hybridization. To control protein loading, rabbit antibodies to β-actin (Abcam, Cambridge, UK, ab227387, 1:5000) were used. All experiments were performed at least three times.

To verify expression of DNMT isoforms in neuronal cultures, we performed immunolabeling for DNMT1 and DNMT3a. After removal of neuronal culture media, cells were washed with PBS and incubated at room temperature for 20 min in freshly prepared 4% paraformaldehyde in PBS. After fixation, cells were washed three times with PBS and neuronal membranes were permeabilized with PBS containing 0.25% Triton X-100 for 15 min at room temperature. Cells were then washed three times in PBS, blocked for 1 h (10% Thermo Blocker bovine serum albumin (BSA) #37525, 0.05% Tween-20, and 300 mM glycine in PBS) and co-incubated with DNMT (1:1,000 in PBS with 10% Thermo Blocker BSA #37525, Abcam anti-DNMT1 (ab87656) or anti-DNMT3a (ab2850)) and MAP2 antibodies (1:250, Anti-MAP2 Alexa Fluor 555 Conjugate, Invitrogen) at 4 °C overnight. Cells were washed three times in PBS and incubated for 1 h at room temperature with a fluorescent secondary antibody (Alexa 488 goat anti-rabbit, Invitrogen; 1:250 in PBS with 10% Thermo Blocker BSA #37525), washed three times with PBS, and mounted onto microscope slides with Prolong Gold anti-fade medium (Invitrogen) containing 4,6-diamidino-2-phenylindole stain as a marker for cell nuclei. Immunostaining experiments were performed in duplicate.