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. 2022 Aug 13;25(9):104923. doi: 10.1016/j.isci.2022.104923

Figure 2.

Figure 2

Identification of mitochondrial targeting sequences (MTS) in STAT6

(A) Schematic of STAT6-WT and domain-deleted mutants with myc-tag. (Δ1–260: 1–260 amino acids-deleted STAT6, ΔDBD: DNA-Binding domain-deleted STAT6, ΔLD: Linker domain-deleted STAT6, ΔSH2: SH2 domain-deleted STAT6, ΔTAD: Transactivation domain-deleted STAT6).

(B) Sucrose-density gradient centrifugation analysis of mitochondria isolated from domain deleted STAT6 mutants-expressing U373-MG stable cell lines. BiP: ER marker.

(C) Sucrose-density gradient centrifugation analysis of mitochondria isolated from 1–50 amino acids-deleted STAT6 (Δ1–50)-expressing U373-MG stable cell line.

(D and E) Confocal images of mitochondria in human U373-MG (D) and mouse B16F10 (E) transfected with GFP or human STAT6 n-terminal fragments with GFP were stained with TOM20. The inset shows a magnification of the selected mitochondrial region (white square). Scale bar, 3μm.

(F and G) At least 18 images/group were quantified for colocalization. Colocalization (Pearson’s Correlation Coefficient) is shown corresponding to the colocalization of the GFP and TOM20 for each group. Data are presented as the means ± SD of 3 independent experiments (∗∗∗p < 0.001). One-way ANOVA with Scheffe’s post-hoc test.

(H) Alignment of the conserved mitochondrial targeting sequences (MTS) identified in STAT6 n-terminal of six mammals. Nucleotide positions that are conserved among the mammals are highlighted with gray rectangle.

(I) Schematic of STAT6-WT and the deletion mutants with myc-tag. (Δ674–847: 674–847 amino acids-deleted STAT6, Δ699–847: 699–847 amino acids-deleted STAT6, and Δ774–847: 774–847 amino acids-deleted STAT6).

(J) PK protection assay of mitochondria isolated from ΔTAD-expressing U373-MG using STAT6 antibody targeted to SH2 domain.

(K) High salt extraction of mitochondria isolated from ΔTAD-expressing U373-MG stable cell lines.

(L) PK protection assay of mitochondria isolated from Δ674-847-expressing U373-MG using STAT6 antibody targeted to SH2 domain.

(M and N) High salt extraction of mitochondria isolated from Δ674–847, Δ699–847, and Δ774-847-expressing U373-MG stable cell lines.