Ab188470

Cells were first lysed (Solarbio, Beijing, China) and then denatured in SDS buffer to obtain total protein. The protein was separated on a 10% SDS-polyacrylamide gel (30 mg per lane) and transferred to a PVDF membrane (Merck Millipore, MA, USA). After blocking in 5% skim milk for 1 h, the membrane was incubated with primary antibody overnight at 4 °C and then incubated with the secondary antibody (1:15,000, GE Healthcare, Cambridge, UK) for 1 h at room temperature. Then, visualization was performed with ECL chemiluminescence reagent (Merck Millipore, MA, USA).
The antibodies used in this study were as follows: CRABP2 (1:1,000, ProteinTech, Cat. # 10225-1-AP), BAX (1:1,000, Abcam, ab32503), BCL-2 (1:1,000, Abcam, ab32124), cleaved caspase-3 (1:1,000, Abcam, ab32042), PARKIN (1:1,000, ProteinTech, Cat. # 14060-1-AP), ATR (1:1,000, Abcam, ab2905), p-ATR (1:1,000, Abcam, ab178407), ATM (1:1,000, Abcam, ab32420), p-ATM (1:1,000, Abcam, ab81292), P53 (1:1,000, Abcam, ab32389), p-P53 (1:1,000, Abcam, ab33889), TET1 (1:1,000, Abcam, ab272900), DNMT3A (1:1,000, Abcam, ab188470), DNMT3B (1:1,000, Abcam, ab2851), P62 (1:1,000, ProteinTech, Cat. # 18420-1-AP), TOMM40 (1:1,000, Abcam, ab185543), α-tubulin (1:5,000, Abcam, ab7291), β-actin (1:5,000, ProteinTech, Cat. # 66009-1-Ig), GAPDH (1:20,000, ProteinTech, Cat. # 60004-1-Ig).

Western blot analysis was performed according to a standard protocol^{65 (link)}. Briefly, whole cells were washed in ice cold PBS before being lysed in RIPA buffer (Thermo Fisher Scientific) containing the protease inhibitor PMSF (Biomed). Next, the proteins in the lysates were separated on SDS-PAGE gels and electrotransferred to PVDF membranes (Millipore, Bedford, MA, USA), followed by blocking in a 5% skim milk solution for 1 h at room temperature under agitation. The membranes were first incubated with primary antibodies and then with HRP-conjugated secondary antibodies. Antibodies were detected using a chemiluminescent HRP substrate (Millipore). Images were obtained using the ImageQuant LAS 500 apparatus (General Electric Healthcare Company, New York, NY, USA). The following antibodies were used: mouse anti-DNMT1 (ab13537, Abcam, Cambridge, UK), rabbit anti-DNMT3a (ab188470, Abcam), rabbit anti-DNMT3b (ab79822, Abcam), mouse anti-GAPDH (ab8245, Abcam), HRP-conjugated rabbit anti-mouse IgG (Sangon Biotech, Shanghai, China P.R.), and HRP-conjugated donkey anti-rabbit IgG (Sangon Biotech).

The paraffin-embedded sections were deparaffinized in xylene and rehydrated. The antigen epitopes were retrieved by microwaving in sodium citrate buffer. Diluted primary antibody DNMT3A (ab188470; 1:2,000; Abcam) was added and incubated for 24 h. After incubating with secondary antibody (ab207999, 1:2,000; Abcam), slides were stained with chromogen diaminobenzidine and then counterstained with hematoxylin. Sections were dehydrated and observed by a microscopy. The number of positive cells was counted in five random areas.

Total proteins from colon tissue and IECs were extracted with RIPA buffer. The lysates were centrifuged at 12,000 × g for 15 min, and the protein concentration was detected with a BCA protein kit (Bio-Rad, USA). Next, the proteins were separated by SDS-PAGE and transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h and incubated with the primary antibodies against anti-occludin (ab216327, abcam, UK), anti-ZO-1(ab96587, abcam), anti-DNMT3a (ab188470, abcam), anti-TNFSF13 (ab108206, abcam), anti-TCAI (ab239370, abcam), and anti-β-actin (ab8226, abcam) at 4°C overnight. Then, the PVDF membrane was incubated with the HRP-conjugated secondary antibody. Finally, the bands were visualized by enhanced chemiluminescence (ECL) kit and gel imaging system.

The colon tissues and IECs were collected and fixed in 4% paraformaldehyde and then performed routinely dewaxing and hydration. Then, the immunofluorescent staining was performed. The sections were incubated with primary antibodies of antioccludin (ab216327, abcam), anti-ZO-1(ab221547, abcam), and anti-DNMT3a (ab188470, abcam) overnight at 4°C. After fully washed with PBST, the sections were incubated with Alexa Fluor 488/594-labeled secondary antibody at room temperature for 1 hour. The nuclei were stained with DAPI (1 μg/mL, Beyotime, China). The images were obtained by a fluorescence microscopy (Nikon, Japan).