Fig 1: Gene regulatory network analysis of SATB2-expressing cells. (A) Scheme of the workflow used to recluster cells after GRN analysis and the resulting tSNE plot of the Satb2 cluster. Regulons were binarized and reclustered accordingly. WT and KO cells segregated based on GRN activity with only minor overlap. (B) Differentially active regulons of the Satb2 cluster that may be possible interactors of TCF4. (C) tSNE plots showing the regulon activity of Ctcf, Cux1, Foxg1, Pou3f3, Smarca4 and Sox11 on a continuous scale (left, red) or binarized (right, blue). The regulons are highly active in the WT cells with only a small number of KO cells showing a high expression. (D) Selection of disease associations enriched in the list of differentially active regulons. (E) Pie charts depicting the percentage of bHLH factors and differentially expressed (DE) regulators in the differentially active regulons. (F) Representative images of TCF4 (white) and FOXG1, POU3F3, SMARCA4 and SOX11 (all in red) immunostaining in the upper third of E18.5 WT cortices. Note the expression in the same nuclei. Scale bars: 50 µm. (G) Co-IP assay using anti-TCF4 antibody conducted with HEK cell extract after overexpression of TCF4 and CUX1, FOXG1, POU3F3, SMARCA4 and SOX11 in HEK cells. The blots presented are cropped. All proteins were co-immunoprecipitated with TCF4, but not with an isotype control for IgG or Agarose A Beads alone except for POU3F3, which was precipitated to a small amount by the isotope control IgG. The interactions were confirmed in three independent biological replicates. ms, antibody raised in mouse. (H) Co-IP assay conducted with E18.5 cortex lysates using anti-SOX11 antibody. Upper panel: detection with anti-TCF4 antibody. Lower panel: detection with anti-SOX11 antibody. The blots presented are cropped. TCF4B was co-immunoprecipitated with SOX11, but not with an isotype control for IgG and Agarose A Beads alone. The interaction was confirmed in three independent biological replicates. rb, antibody raised in rabbit.
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