Proximity biotinylation captures the proteins near Foxp3. (A) Schematic of the PSI of Foxp3 by proximity ligation via biotin-phenoxyl radicals. 1° Ab, primary antibody; 2° Ab, secondary antibodies; BP, biotin-phenol; HRP, horseradish peroxidase; SA, streptavidin. (B) Assessment of the specificity of Foxp3 PSI with Th0 and iTreg cells. CD4 Tn cells from Foxp3^gfp mice were used to generate Th0 or iTreg cells. After PSI reaction, cells were stained with SA-AlexaFluor-568. Numbers show median fluorescence intensities (MFIs) of GFP or SA in Th0 and iTreg cells. Th0 cells, CD4 Tn cells activated by TCR agonists and IL-2 in vitro. (C) Immunofluorescence images of Treg cells from Foxp3^gfp mice treated with Foxp3-PSI and subsequently stained for SA-AlexaFluor-568, GFP-booster-FITC, and Lamin-AlexaFluor-647. (D) 37,333 accessible regions (ATAC-seq) in Treg cells are cross-compared with Foxp3 PSI-ChIP, traditional (tra) Foxp3-, H3K27ac-, H3K4me3-, and H3K27me3-ChIP peaks. Traditional Foxp3, H3K27ac, H3K4me3, and H2K27me3 ChIP-seq data are from Kitagawa et al. (2017). (E) Comparison of accessible chromatin regions (ATAC-seq peaks) in iTreg and nTreg cells. (F) Comparison of the peak sizes of Foxp3 PSI-ChIP, traditional Foxp3-ChIP, H3K27ac-ChIP, and ATAC-seq. Numbers show mean fragment lengths. Data were derived from two replicates. (G) PCA of Foxp3 PSI and histone H3 PSI TMT MS results. PC1 and PC2, respectively, account for 48.9% and 27.7% of the total variations. AU, arbitrary units. (H) Fractions of proteins identified by Foxp3 or H3 PSI MS among the genes expressed in ASC-treated iTreg cells, estimated by RNA-seq (RPKM ≥ 1.0). (I) Enriched proteins identified by Foxp3-PSI in iTreg versus Th0 cells and Foxp3-PSI versus H3-PSI in iTreg cells. Blue, Foxp3 known interactors. q, FDR-adjusted P value. Histone H3 PSI was used to assess the diffusion of BP radicals. Data were derived from two replicates. (J) Comparison of proteins revealed by Foxp3-PSI versus H3-PSI MS and published Foxp3 interactors (Rudra et al., 2012).