Intracellular distribution of α-TAT1 mediates its function. (a–i) (a) Cartoon showing α-TAT1 with predicted NES and NLS in intrinsically disordered C-terminus, adapted from PDB accession no. 4GS4; (b) intracellular distribution of mVenus-α-TAT1; red dotted lines outline nuclei as identified by H2B-mCherry (cytosolic and diffused) or DAPI (nucleus enriched) in lower panel; (c) categorical analysis (318 cells; Nuc: Nucleus enriched, Diff: Diffused, Cyto: Cytosolic); (d) ratiometric analysis (184 cells) and (e) colocalization analysis (180 cells) of mVenus-α-TAT1 localization in HeLa cells; (f) immunostaining with anti–α-TAT1 antibody showing intracellular localization of endogenous α-TAT1 in MEF cells; red dotted lines outline nuclei as identified by DAPI; (g) cartoon showing α-TAT1 mutants used in (h) immunofluorescence assays showing levels of acetylated α-tubulin and total α-tubulin, transfected cells are indicated with red arrowheads; (i) ratio of acetylated α-tubulin to total tubulin intensities with exogenous expression of α-TAT1 and its mutants, normalized against that of non-transfected cells (WT: 50, catalytic domain: 44, NLS-catalytic domain: 48 cells). Scale bar = 10 µm. ***, P < 0.001 or as shown, Student’s t test.