Fig 1: Physical interaction between SMAD1 and KDM1A. A, coimmunoprecipitation of Myc-tagged SMAD1 with the indicated FLAG-tagged coregulators in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. B, in vitro pulldown of KDM1A (0.5 μg) and/or SMAD4 (0.5 μg) with SMAD1 (0.5 μg) as bait. Blots are representative of n = 3 independent biological replicates. Right, quantification of band intensity is shown on the right. Results of n = 3 independent biological replicates are presented as scatter plots with bar graphs, which indicate mean ± SD. Differences between the conditions were analyzed by Tukey's honestly significant difference test corrected for multiple comparisons. C, coimmunoprecipitation of endogenous SMAD1/5 protein with KDM1A in WT mESCs. SMAD1/5 immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. Right, quantification of each lane of the blots. Results of n = 3 independent biological replicates are presented as scatter plots with bar graphs, which indicate mean ± SD. Differences between the conditions were analyzed by Tukey's honestly significant difference test corrected for multiple comparisons. D and E, schematic illustrations of interaction between SMAD1 and KDM1A. F, coimmunoprecipitation of HA-tagged KDM1A with the indicated FLAG-tagged SMAD1 mutants in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. G, coimmunoprecipitation of HA-tagged KDM1A with the indicated FLAG-tagged MH2 domain of SMAD protein or IAD of IRF3 in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. H, coimmunoprecipitation of Myc-tagged SMAD1 with the indicated FLAG-tagged KDM1A mutants in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. I, coimmunoprecipitation of Myc-tagged SMAD1 with the indicated FLAG-tagged KDM1A mutants in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. ∗, ∗∗, ∗∗∗∗, and ns represent p < 0.05, 0.01, 0.0001, and not significant, respectively. HEK293T, human embryonic kidney 293T cell line; mESC, mouse embryonic stem cell; MH2, Mad Homology 2 domain.
Fig 2: Involvement of KDM1A/LSD1 in regulation of the genes downregulated by SMAD1/5. A, a schematic illustration of candidate models governing the transcriptional regulation of S15down genes (1). SMAD1/5 colocalize with factor X that demethylates H3K4 or (2) SMAD1/5 competes with factor X that methylates H3K4. B, a Venn diagram indicating the overlap of proteins preferentially bind to the gene loci of 40 DEGs (1027 datasets or 139 proteins) and proteins colocalized with SMAD1 on a genome-wide scale (482 datasets or 135 proteins). C, a list of 20 proteins depicted within the overlapped region in Figure 3B. D and E, results of preranked gene set enrichment analysis (GSEA) with the custom gene set of the S15down genes (20 genes) in mESCs deficient in indicated gene(s) compared with control cells. D, a table presents the p value and enrichment score (ES) of GSEA. A negative ES indicates that members of the gene set tend to appear at the bottom of the ranked transcriptome data or highly expressed in mESCs deficient in indicated gene(s). E, GSEA plots showing the enrichment results of the custom gene set in Kdm1a-deficient mESCs of two different studies. F, MA plots of DESeq2 data of Kdm1a-deficient mESCs, showing log2 fold change (M, on the y-axis) against the mean of the normalized counts (A, on the x-axis). Circles colored light blue indicate genes whose transcript abundance were significantly altered (adjusted p < 0.05) based on both DESeq2. G, qRT–PCR analysis of mRNA of indicated genes in mESCs treated with KDM1A/LSD1 inhibitors, DMH1, or DMSO as control. Actb was used as endogenous control. Results of n = 3 independent biological replicates are presented as scatter plots with bar graphs, which indicate mean ± SD. Differences between the conditions were analyzed by Tukey's honestly significant difference test corrected for multiple comparisons. ∗, ∗∗, ∗∗∗, ∗∗∗∗, and ns represent p < 0.05, 0.01, 0.001, 0.0001, and not significant, respectively. DEG, differentially expressed gene; DMSO, dimethyl sulfoxide; mESC, mouse embryonic stem cell; qRT–PCR, quantitative RT–PCR.
Supplier Page from Abcam for Recombinant Human Smad1 protein