Dnmt1

Dnmt1, Dnmt3a, Cdc25A and GAPDH antibodies were purchased from GeneTex (Hsinchu city, Taiwan). Bcl-2, Bcl-xL, Cdk6, Dnmt3b, and p53 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The c-Myc, sGCα1 and CyclinD1 antibodies were purchased from Epitomics/Abcam (Cambridge, UK). The sGCβ1 antibody was purchased from Calbiochem/Millipore (Billerica, MA, USA). Cyclin B1 antibody was purchased from Upstate Biotechnology/Millipore (Lake Placid, NY, USA). Cdc2 and Skp2 antibody were purchased from Santa Cruz (Dallas, TX, USA).

The cells were lysed in RIPA buffer for 30 min and then centrifuged at 13,000 rpm for 15 min at 4 ^oC. The membrane/cytoplasmic protein fractions of the cultured cells were obtained using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo, Waltham, MA, USA). The protein concentration was measured using a BCA protein assay (Thermo, Waltham, MA, USA). Total proteins (30 µg) were separated by SDS-PAGE on 10% polyacrylamide gels and transferred to PVDF membranes. The membranes were hybridized with primary antibodies overnight after blocking for 30 min in 5% nonfat milk. Immunoblotting was performed with primary antibodies against DNMT1 (GeneTex, Hsinchu City, Taiwan), p-Akt (Cell Signaling Technology, Danvers, MA, USA), Akt (Cell Signaling Technology, Danvers, MA, USA), HTR2B (GeneTex, Hsinchu City, Taiwan), ALDOC (Abcam, Cambridge, UK), PPARγ (Abcam, Cambridge, UK), PTGS2 (GeneTex, Hsinchu City, Taiwan), NR2F1 (GeneTex, Hsinchu City, Taiwan) and β-actin (Sigma, St. L ouis, MO, USA). A chemiluminescence system was used to visualize the immunoreactive bands (Amersham ECL PlusTM, GE Healthcare Life Sciences, Chalfont St. Giles, UK).

Fetal mouse brain tissues were fully fixed with 4% paraformaldehyde. After 48 h, these tissues were washed by PBS, dehydrated by ethanol, paraffin-embedded, sliced and washing again. Subsequently, these tissues were dissolved in ethanol and washing. The tissue antigen was repaired. Blocking was using 5% goat serum. TET1 (dilution 1:100; Genetex), TET2 (dilution 1:100; Genetex), TET3 (dilution 1:100; Genetex), DNMT1(dilution 1:100; Genetex), DNMT3b (dilution 1:100; Genetex) as the primary antibodies was performed at 4 °C overnight. Quality was assessed on each batch of slides including a negative control. And the primary antibody was substituted by 10% normal goat serum in order to rule out non-specific signals.