Antibodies against 5 hmc

The GES‐1 cell line was provided by the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The AGS, SGC‐7901, and SK‐GT5 cell lines were purchased from ATCC. SGC‐7901 and SK‐GT5 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) and an environment of 5% CO₂ at 37°C, and GES‐1 cells were cultivated in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 10% fetal bovine serum (Life Technologies/Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin (Life Technologies/Gibco). The plasmids of αSR and CagA‐wild type were purchased from Dr Masanori Hatakeyama of Japan's Tokyo University. The pEGFP‐N1 plasmid was purchased from Clontech, the plasmids TET1‐wt and pX459 were obtained from Addgene, and pX459‐TET1‐sgRNA was constructed in the laboratory. The liposome transfection reagent Lipofectamine 3000 was purchased from Invitrogen in the United States. Antibodies against HA were purchased from OriGene, antibodies against CagA were purchased from Santa Cruz, antibodies against 5‐hmC were purchased from Active Motif, and antibodies against KLF4 and TET1 were purchased from Santa Cruz.

Antibodies against 5-hmC (Active Motif, Carlsbad, CA, USA) were used as primary antibodies. The secondary antibody used was a ready-for-use anti-rabbit EnVision-Peroxidase system (Dako Japan Inc., Tokyo, Japan). Nuclear 5-hmC expression was recorded as negative, weak, or strong expression by an investigator (Y.B.) unaware of other data. 5-hmC-positive expression was defined as weak or strong expression in this study