Dnmt3a

We homogenized mouse tissues in buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P‐40, 0.5% deoxycholate, 0.1% SDS, and 1 mM 2‐mercaptoethanol with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, NJ, USA) and then centrifuged them at 2,500 × g for 15 min. Equal amounts of protein were separated by 5–20% SDS–PAGE and transferred to Hybond‐P membranes (GE Healthcare, Piscataway, NJ, USA). The primary antibodies and their dilutions were as follows: AR (N20, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Dnmt1 (1:1,000; Abcam, Cambridge, MA, USA), Dnmt3a (1:1,000; Abcam), Dnmt3b (1:1,000; Abcam), Hes5 (1:1,000; Santa Cruz), and ChAT (1:1,000; Millipore, Billerica, MA, USA). Primary antibody binding was probed with horseradish peroxidase‐conjugated secondary antibodies at a dilution of 1:5,000, and bands were detected by using an immunoreaction enhancing solution (Can Get Signal; Toyobo, Osaka, Japan) and enhanced chemiluminescence (ECL Prime; GE Healthcare). An LAS‐3000 imaging system (Fujifilm, Tokyo, Japan) was used to produce digital images. The signal intensities of these independent blots were quantified using IMAGE GAUGE software version 4.22 (Fuji) and expressed in arbitrary units (n = 3 for each group). The membranes were reprobed, or the same samples were examined with an anti‐GAPDH (1:5,000; Santa Cruz) antibody for normalization.

Western blotting was performed according to standard procedures using enhanced chemiluminescence detection. Briefly, total proteins were extracted from cells using RIPA lysis buffer (Thermo Fisher Scientific). Equal amounts of protein were separated on sodium dodecyl sulfate–polyacrylamide gels and then polyvinylidene fluoride membrane (Millipore). After blocking with 5% skim milk, the membranes were incubated with the primary antibodies for DNMT3A, DNMT3B, E-cadherin, N-cadherin, vimentin and GAPDH (Abcam), followed by incubation with corresponding horseradish peroxidase-coupled secondary antibody (Beyotime). The signals were detected with enhanced chemiluminescence reagents (Pierce). GAPDH was used as an internal control.