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[Preprint]. 2025 May 3:2025.05.02.651992. [Version 1] doi: 10.1101/2025.05.02.651992

Figure 6. Identification of protein components of TEAD1 foci with proximity-based mass spectrometry.

Figure 6.

(A) Schematic of the proximity labeling experimental setup for protein identification in TEAD1 foci. mT is short for miniTurbo.

(B) Representative immunofluorescence images of UOK342 transiently expressing miniTurbo-TEAD1 WT-HaloTag (WT) and miniTurbo-TEAD1 H3 mutant-HaloTag (H3Mut) showing biotinylation (red) in relation to HaloTag (magenta), TEAD1 (green), and nuclei (blue). Scale bars represent 5 μm.

(C) Venn diagram summarizing unique and shared biotin-peptides identified in mass spec between TEAD1 WT and TEAD1 H3Mut.

(D) Volcano plots showing differentially enriched biotinylated proteins comparing WT / H3Mut (n=3 WT and n=3 H3Mut). Red symbols (two-sided t test unadjusted p ≤ 0.05 and ≥ 2-fold change) represent biotinylated proteins enriched in cells expressing miniTurbo-TEAD1 WT-HaloTag, and blue symbols represent biotinylated proteins enriched in cells expressing miniTurbo-TEAD1 H3Mut-HaloTag.

(E) Results from GSEA of ≥ 2-fold biotinylated proteins in miniTurbo-TEAD1 WT-HaloTag showed enrichment of chromatin remodeling and negative regulation on transcription.

(F) Representative images of TEAD4 colocalizing with TEAD1 foci and HMGB2 colocalizing with the nucleolar-localized TEAD1 foci. Scale bars represent 5 μm.

(G) Quantification of co-localization of TEAD1 with TEAD4 (left) and HMGB2 (right) in UOK342 cells around TEAD1 foci (foci) versus another random nuclear region (control). Mean ± SEM, ***, p ≤ 0.0005; ****, p ≤ 0.0001. Unpaired t-test.