Ab3493

Protein was extracted using lysis buffer and quantified by Pierce™ BCA Protein Assay Kit (Thermo, 23 227). 1 μL PMSF (0.1 mol/L) and 10 μL phosphatase inhibitor (10 g/L) were added per 1 mL lysis buffer immediately before use. Denatured proteins were resolved using 10%–12% SDS‐PAGE gels and blotted on PVDF membranes. Following blocking in 5% skim milk/0.2% Tween/dH₂O, membranes were incubated with primary antibodies at 4°C for overnight. The primary antibodies used were anti‐ZSCAN4 (Abcam, ab106646; dilution 1:1000), anti‐DNMT3A (CST, 3598S; dilution 1:1000), anti‐DNMT3L (Abcam, ab3493; dilution 1:2500), anti‐DNMT1 (Abcam, ab19905; dilution 1:1000), anti‐β‐CATENIN (CST, 8480; dilution 1:1000), anti‐H3K27ac (Abcam, ab4729; dilution 1:1000), anti‐H3 (Abcam, ab176842; dilution 1:1000) and anti‐β‐ACTIN (Abcam, ab8227; dilution 1:5000). Horseradish peroxidase‐conjugated secondary antibodies (1:5000) were incubated for 1 hours at room temperature, and proteins were detected by ECL plus reagent. After rinsing with TBST, Clarity™ Western ECL Substrate (BIO‐RAD) was used for visualization, and ChemiDoc™ MP Imaging System (BIO‐RAD) was used for band detection.

Cells were collected with trypsin, washed 3 times with PBS, and lysed in buffer that contained 20 mM Tris (pH 8.0), 137 mM NaCl, 100 g/L glycerol, 50 g/L Triton X-100, and 4 g/L EDTA; 1 μl PMSF (0.1 M) and 10 μl phosphatase inhibitor (10 g/L) were added per 1 ml lysis buffer immediately before use. Proteins were denatured with 2 × SDS at 95°C for 5 min. A total of 20 μg denatured protein was run on 8% or 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. Membranes were blocked with 5% nonfat milk in 1 × TBS with 0.05% Tween 20 (TBST) for 1 h. Samples were probed with primary antibodies overnight at 4°C. The primary antibodies used were anti-DNMT3A (CST, 3598S; dilution 1:1,000), anti-DNMT3B (Abcam, ab78922; dilution 1:2,000), anti-DNMT3L (Abcam, ab3493; dilution 1:2,500), anti-DNMT1 (Abcam, ab19905; dilution 1:1,000), and anti-β-ACTIN (Abcam, ab8227; dilution 1:5,000). Blots were rinsed with TBST. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 60 min at room temperature, and proteins were detected by ECL plus reagent. After rinsing with TBST, Clarity Western ECL Substrate (Bio-Rad) was used for visualization, and ChemiDoc MP Imaging System (Bio-Rad) was used for band detection.

Total protein extracts were prepared from tissues and cells using RIPA buffer (Cell Signaling). Equivalent amounts of protein determined by BCA assay were resolved by 10% Bis-Tris gel electrophoresis and blotted to Immobilon PVDF membranes (Millipore). The following primary antibodies and dilutions were used: anti-DNMT1 (1:1000; ab13537, Abcam); anti-DNMT3a (1:1000; ab188470, Abcam); anti-DNMT3b (1:1000; ab79822, Abcam); anti-DNMT3L (1:1000; ab3493, Abcam); anti-BMP4 (1:1000; ab39973, Abcam); anti-TBX3 (1:1000; ab154828, Abcam); anti-ACADM (1:1000; ab92461, Abcam); anti-caspase 3 (1:1000; 9662S, Cell Signaling); anti-GAPDH (1:5000; 5174S, Cell Signaling). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Proteintech) was used as secondary antibody. The membranes were developed using Supersignal West Pico Chemiluminescent substrate (Thermo Scientific). To normalize sample loading and transfer, the ratio of band intensities to GAPDH was obtained to quantify the relative protein expression level.