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. 2015 Jan 21;11(1):775. doi: 10.15252/msb.20145504

Figure 5.

Figure 5

Functional validation of FOXN2 based on its interacting proteins in soluble and chromatin fractions
  1. FOXN2 HCIPs form distinct complexes in chromatin versus soluble fractions. The size of prey dots indicates the estimated abundance of preys. Lines indicate the interactions defined in the literature. CUL1 was identified in a parallel virus-based FOXN2 purification.
  2. 293T cells were transfected with constructs encoding MYC-tagged RFX1 and SFB-tagged FOXN2 or its DNA binding-defective mutant FOXN2 (H162R) as indicated. Pull-down experiments were carried out with S-protein beads and immunoblotted with antibodies as indicated.
  3. Overlap Venn diagram of FOXN2 and RFX1 target genes identified by ChIP-sequencing. 293T cells stably expressing SFB-tagged FOXN2 or RFX1 were subjected to ChIP-sequencing using anti-FLAG antibody. Each experiment was performed with two biological replicates, and four control ChIP-sequencings were performed using 293T cells stably expressing other TFs.
  4. Reverse purification of FBXW11 (βTRCP2)-containing protein complexes conducted using the same TAP/MS protocol recovered FOXN2 as FBXW11-binding protein. Prey names, peptide counts and whether or not the interactions have been reported were listed.
  5. 293T cells were transfected with constructs encoding SFB-tagged FOXN2 and MYC-tagged βTRCP, its substrate binding-defective mutant βTRCP (R474A), or βTRCP2 as indicated. Pull-down experiments were carried out with S-protein beads and immunoblotted with antibodies as indicated.
  6. In vivo ubiquitination assays were performed by co-transfecting constructs encoding FLAG-tagged FOXN2, His-tagged ubiquitin, MYC-tagged βTRCP, βTRCP (R474A) or βTRCP2 into HEK293T cells as indicated. Cell lysates were denatured with 1% SDS and diluted 10-fold using PBS prior to the pull-down by Ni-NTA resin, followed by immunoblot with antibodies as indicated.
  7. 293T or 293T-shβTRCP2, 293T-shβTRCP2, 293T-shCUL1 cells were treated with 100 mM cycloheximide (CHX) for the indicated time. Immunoblotting was conducted with antibodies as indicated.
  8. A model showing on/off chromatin regulation of FOXN2 by transcriptional co-factors or E3 ligase complexes. All of the components indicated were identified from FOXN2 purifications.

Source data are available online for this figure.