Fig. 2: PHF14 is identified as a novel binding partner of DNMT3B.
From: PHF14 enhances DNA methylation of SMAD7 gene to promote TGF-β-driven lung adenocarcinoma metastasis
a Putative bound proteins of DNMT3B were analyzed by IP-MS assay. Representative image was derived from 3 independent experiments. b, c Immunoprecipitation assays with Flag-tagged PHF14 and HA-tagged DNMT3B, respectively, in LAD cells and 293FT cells validated an interaction between PHF14 and DNMT3B. Incubation of IgG-conjugated beads with cell lysates was employed as the negative control in these assays. Representative blots were derived from 3 independent experiments. d WB analysis showed the effect of overexpressing (left panel) or knocking out PHF14 (right panel) on the protein level of SMAD7 in indicated LAD cells. e qPCR analysis showed the effect of overexpressing PHF14 on the mRNA level of SMAD7 in Calu3 and HCC827 cells with indicated treatments. f BSP validated the DNA methylation level of region 1 of SMAD7 gene locus in Calu3 and HCC827 cells with indicated treatments. g Measurement of SBE-luciferase activity showed relative TGF-β signaling activates in Calu3 and HCC827 cells with indicated treatments. h qPCR analysis showed the effect of knocking out PHF14 on the mRNA level of SMAD7 in A549 and PC9 cells with indicated treatments. i BSP validated the DNA methylation level of region 1 of SMAD7 gene locus in A549 and PC9 cells with indicated treatments. j Measurement of SBE-luciferase activity showed relative TGF-β signaling activates in A549 and PC9 cells with indicated treatments. For aforementioned WB, qPCR, BSP and SBE-luciferase activity assays, cells were treated with TGF-β1 at final concentration of 5 ng/mL for 48 h before the indicated assays were performed (d–j). Error bars represent means ± SD derived from three independent experiments. Two-way ANOVA multiple comparison analysis was used for statistical analysis. **P < 0.01; ns, not significant, P > 0.05.
