TET3 Antibody - #DF13335

FIGURE 2 The promoter of IGF2BP3 was hypomethylated in triple-negative breast cancer (TNBC). (A) Schematic representation of the CpG islands in the IGF2BP3 promoter. The red region is the input sequence; the blue region is CpG islands. (B) Promoter methylation level of IGF2BP3 in different molecular subtypes of breast cancer tissues from TCGA dataset. (C) Methylation-specific PCR of the CpG island of the IGF2BP3 promoter region in different breast cancer cell lines and matched normal breast cell line. (D and E) qRT-PCR was used to confirm IGF2BP3 expression at the mRNA level. (F) Western blotting was used to confirm the IGF2BP3 and TET3 expression at the protein level. Data are shown as the mean ± SEM; *p < .05. (G) Methylation-specific PCR of the CpG island of the IGF2BP3 promoter region in TET3 knockdown MDA-MB-231 and HCC-1806 cells.

Fig 7. The substrate binding domain of SPHK2 interacts with TET3 and HDAC1 and inhibits IFN-β transcription. (A) A549 cells transfected with pCMV-Myc or Myc-SPHK2 were immunoprecipitated using anti-Myc antibody, and then Myc-tagged SPHK2, HDAC1, and TET3 proteins were analyzed by immunoblotting. (B) A549 cells transfected with pRK11-Flag or Flag-TET3 were immunoprecipitated using anti-Flag antibody, and then SPHK2, HDAC1, and Flag-tagged TET3 proteins were analyzed by immunoblotting. (C) A549 cells transfected with pCMV-Myc or Myc-HDAC1 were immunoprecipitated using anti-Myc antibody, and then SPHK2, Myc-tagged HDAC1, and TET3 proteins were analyzed by immunoblotting. (D) A549 cells uninfected or infected with WSN were immunoprecipitated using anti-Human SPHK2 polyclonal antibody (Proteintech, 17096-1-AP), and then SPHK2, HDAC1 and TET3 proteins were analyzed by immunoblotting. (E) Diagram of SPHK2-FL (full length) and deletion of SPHK2 fragments (F1, F2 and F3). (F) A 549 cells transfected with pCMV-Myc, Myc-SPHK2-FL, Myc-SPHK2-F1, Myc-SPHK2-F2, and Myc-SPHK2-F3 were immunoprecipitated by using anti-Myc antibody, and then Myc-tagged SPHK2, Myc-tagged SPHK2-F3, HDAC1, and TET3 proteins were analyzed by immunoblotting. (G) HEK293 cells were transfected with pCMV-Myc, Myc-SPHK2-FL, Myc-SPHK2-F1, Myc-SPHK2-F2, or Myc-SPHK2-F3, and then infected with WSN virus at an MOI of 0.5. IFN-β activity was measured by using luciferase reporter assays. (H) A549 cells transfected with pCMV-Myc (Mock), Myc-SPHK2 or Myc-SPHK2-F3, and IFN-β promoter enrichment for Myc-tagged SPHK2 or Myc-tagged SPHK2-F3 was detected by ChIP-qPCR. (I) IFN-β promoter enrichment for Myc-tagged HDAC1 in sgCTRL A549 cells or sgSPHK2-1 A549 cells overexpressed with Myc-tagged HDAC1 was detected by ChIP-qPCR. (J) A549 cells were transfected with CTRL-siRNA or TET3-siRNA, and 24 h post-transfection, cells were overexpressed with SPHK2. IFN-β promoter enrichment for Myc-tagged SPHK2 was detected by ChIP-qPCR. (K) A549 cells were uninfected or infected with WSN, and IFN-β promoter enrichment for SPHK2 was determined by ChIP-qPCR. Data are representative of three independent experiments. ns, no significance; **, P