Anti tet1

Representative areas were selected via H&E staining, and anti‐TET1 (Abcam, Shanghai, China) or anti‐Twist was used for IHC according to manufacturer's programme.

The isolated left temporal lobe cortex was added with ice-cold protein lysate and centrifuged at 12000 r/min for 20 min at 4°C. Protein samples were separated by electrophoresis using SDS-PAGE and transferred onto PVDF membranes (Millipore, United States). After blocking with 5% nonfat milk for 2 h at room temperature, the membranes were incubated with the primary antibodies at 4°C overnight and then incubated with HRP-conjugated secondary IgG antibodies. The primary antibodies used here included anti-Tet1 (1 : 1000, Abcam, UK, Cat. No. 191698), anti-Tet2 (1 : 1000, Millipore, United States, Cat. # ABE364), anti-Slc44a1 (1 : 1000, Abcam, UK, Cat. No. 110767), and anti-β tubulin (1 : 2000, Cell Signaling Technology, United States, Cat. #2128). The membranes were then developed using ECL detection, and the signals were detected with Tanon-5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). The protein levels were analyzed by Image J software (NIH, Bethesda, USA) and normalized to the corresponding β-tubulin level.

To verify whether transfection with pSELECT-GFPzeo-TET1 or pSELECT-GFPzeo-TET2 vectors results in the production of the respective TET proteins, HEK293 cells were suspended in 500 µL of RIPA buffer containing protease inhibitor mix, incubated on ice for 10 min, and then centrifuged at 400× g for 20 min at 4 °C. The protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Twenty micrograms of the extract were used for electrophoresis and transferred to the membrane following standard procedures. The membrane was blocked and incubated with anti-TET1 (1:1000; Abcam, Cambridge, UK), anti-TET2 (1:500, Active-Motif, Carlsbad, CA, USA), or anti-GFP (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) primary antibodies, washed in TBST and incubated with the appropriate secondary antibodies. Signals were detected using the Clarity ECL Western Blotting Substrate (Bio-Rad, Hercules, CA, USA) and analyzed using a GeneGnome chemiluminescence imaging system (Syngene, Cambridge, UK) and Image Studio Lite 4.0 software (LI-COR, Lincoln, NE, USA) (Supplementary Figure S1).

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 solution for 20 min. Cells were washed with PBS and then blocked in 5% bovine serum albumin for 1 h at room temperature. Coverslips were incubated with anti-ADCY6 (1:300, Abcam, USA) and anti- TET1 (1:500, Abcam, USA) antibodies overnight at 4°C. After washing with PBS, cells were incubated with appropriate secondary antibody and 4′,6-diamidino-2-phenylindole (DAPI). Slides were imaged using an inverted fluorescence microscope.