Ab122932

Total protein extracts were prepared using a lysis buffer (50 mM Tris, 2% SDS, 10% glycerol, 0.74 M beta-mercaptoethanol), sonicated on ice using a Sonifier 250D (Branson Ultrasonics), and heated for 5 min at 99 °C. Protein concentrations were determined with the Bio-Rad DC Protein Assay Kit (BioRad) using bovine serum albumin as a standard. Protein extracts were separated in 10% SDS-PAGE and electrotransferred to PVDF membranes (Immobilon-P; EMD Millipore). Antibodies used were as follows: MYC #ab32 (Abcam, Cambridge, USA) [56 (link)], #sc764 (Santa Cruz Biotechnology, USA) [57 (link)]; DNMT3B #ab122932 (Abcam, Cambridge, USA) [58 (link)],#nb100-56514 (Novus Biologicals, USA) [59 (link)]; TUBULIN #ab6046 (Abcam, Cambridge, USA) [22 (link)]; β-ACTIN #A5441 (Sigma-Aldrich, USA); GAPDH #sc25778 (Santa Cruz Biotechnology, USA) [60 (link)]; Anti-MOUSE HRP #31430 (Thermo Fisher, USA) [61 (link)]; Anti-RABBIT HRP #31460 (Thermo Fisher, USA) [62 (link)]. Protein quantitation was performed using ImageJ software (https://imagej.nih.gov/ij/) [63 (link)].

Proteins were extracted by resuspending the pellet of cells from a 20 ml cultures at OD600 = 1 in 400 µl of RIPA buffer (50 mM Tris pH7.5, 150 mM NaCl, 1% NP40, 0.5% NaDeoxycholate, 0.1% SDS) containing 1 mM PMSF and protease inhibitors (cOmplete ULTRA Tablets, Mini, EASYpack, Roche). 400 µl of glass beads were added and samples were processed using FastPrep (MP) for 3 times for 20 s pulses @4.5 m/s. After centrifugation 5 min at 2350 x g, supernatants were recovered and quantified by bradford. 20 µg of protein were loaded on a 6 or 8% acrylamide gel and subjected to PAGE, proteins were then transferred onto an immobilon membrane (millipore) for subsequent hybridization with anti-DNMT1 (ref ab87654, Abcam, dilution 1/1000), anti-DNMT3a (ab2850, Abcam, dilution 1/2000), anti-DNMT3b (ab122932, Abcam, dilution 1/500) or anti-Flag (F7425, Sigma, dilution 1/2000) antibody overnight followed by secondary antibody anti Rabbit (Goat)-HRP conjugated (65-6120, Invitrogen, dilution 1/5000). The signal was revealed using ECL^TM prime WB detection reagent (Amersham, GE Healthcare).

5 × 10⁵ mESCs were harvested and lysed in Laemmli sample buffer. 10 μg of protein extracts were separated on NuPAGE 4–12% Bis-Tris protein gels (Invitrogen) and blotted to nitrocellulose membrane (Millipore). The membranes were probed with primary antibodies against DNMT3A (sc-365769; Santa Cruz), DNMT3B (ab122932; Abcam), and Tubulin (sc-58880; Santa Cruz), and corresponding secondary antibodies (680RD/800CW anti-mouse IgG, and anti-rabbit IgG; RDye). The membranes were scanned by an Odyssey Infrared Imager, and analyzed by the software Image Studio.