Journal: STAR Protocols

Article Title: Protocol for mapping murine transcription factor interactomes and composite motifs combining affinity purification mass spectrometry and ChIP-seq

doi: 10.1016/j.xpro.2025.104184

Figure Lengend Snippet: Generation of the IRF4 Bio mouse strain and in vitro differentiation of primary T cells into Th17 cells (A) Schematic overview of the bacterial artificial chromosome (BAC) transgene production for the generation of the IRF4 Bio mouse strain. (B) Cross-breeding of animals: IRF4 Avi mice, containing the BAC transgene, are crossed with ROSA26 BirA mice, ubiquitously expressing BirA ligase under the ROSA26 promoter, resulting in offsprings that express in vivo biotinylated IRF4 (IRF4 Bio ). (C) Western blot analyses of full cellular lysates and nuclear extracts demonstrate successful in vivo biotinylation of IRF4 (left, anti-IRF4 antibody; right, horseradish peroxidase-conjugated streptavidin [SA-HPO]). (D) Workflow for in vitro differentiation of primary Th17 cells.

Article Snippet: For example, in the case of the TF IRF4, good results were achieved using the μMACS FactorFinder Kit by Miltenyi, which has been discontinued.

Techniques: In Vitro, Expressing, In Vivo, Western Blot

Journal: STAR Protocols

Article Title: Protocol for mapping murine transcription factor interactomes and composite motifs combining affinity purification mass spectrometry and ChIP-seq

doi: 10.1016/j.xpro.2025.104184

Figure Lengend Snippet: Integrated analysis of IRF4 interactors and double-motif occurrence (A) Double-motif occurrence of IRF4 interactors in Th17 ChIP-seq peaks. Heatmap shows normalized co-occurrence frequencies (0–1) for IRF4 together with transcription factors detected by AP-MS, considering only motifs occurring within <5 bp spacing in the same peak. Asterisk (∗): GTF2IRD1-isoform2. (B) Volcano plot of IRF4 interactors identified by AP-MS comparing biotinylated IRF4 (“Bio”) and control (“Ctrl”) conditions in Th17 cells. Labeled interactors indicate TFs with double-motif occurrences in Th17. (C) Sequence logos of the individual TF motifs used for double-motif annotation. (D) STRING-based IRF4 interaction network displaying interactors classified as TFs according to the criteria by Lambert et al. (STRING DB default settings). See also .

Article Snippet: For example, in the case of the TF IRF4, good results were achieved using the μMACS FactorFinder Kit by Miltenyi, which has been discontinued.

Techniques: ChIP-sequencing, Protein-Protein interactions, Control, Labeling, Sequencing