Fig. 4.

αTAT1 catalytic activation requires direct phosphorylation at Ser237 by TAK1. a Schematic representation of αTAT1 serine/threonine to alanine single point mutants. b COS-7 cells expressing αTAT1 WT or the serine/threonine to alanine single point mutants were fixed and imaged for αHA and Acetyl-tubulin. CTCF was quantified using Image J, at least 25 cells were quantified per condition. The images are representative of three independent experiments, n = 3. Error bars represent SEM and type 2 t-test analysis show relative to αTAT1 WT: *p < 0.001. c αTAT1^–/– MEFs expressing TAK1 WT or HA-αTAT1 WT, HA-αTAT1-S237A, and HA-αTAT1-S237E were fixed and imaged for Acetyl-tubulin (red), αHA (pseudo color cyan), and TAK1 (green) immunofluorescence co-staining. CTCF per unit area was quantified using Image J, at least 30 cells were quantified per condition. Error bars represent SEM and type 2 t-test analysis show relative to αTAT1 WT: *p < 0.001; **p < 0.0001. d COS-7cells expressing HA-αTAT1 WT or HA-αTAT1-S237A were subjected to nocodazole (100 nM) for 1 h, washed thoroughly with PBS, and then immediately fixed after 0, 15, 30, 45, 60, 90, and 120 min. The fixed cells were then imaged for αHA and Acetyl tubulin immunofluorescence co-staining. The images were quantified by skeletonizing the acetyl tubulin staining using Image J and skeletons analyzed for length and branching of acetyl tubulin filaments. e αTAT1^+/+ MEFs, αTAT1^–/– MEFs, and mouse embryonic endothelial cells were treated with TGF-β (200 pM), LPS (10 ng/ml) for 30 min, or pre-treated with 5Z-7-oxozeaenol (20 μM) for 15 min followed by TGF-β stimulation. f In vitro kinase assay performed using purified αTAT1, TAK1, and TAB1 were incubated with and without treatment with 5Z-7-oxozeaenol (20 μM). g In vitro acetylation assay performed using purified tubulin, αTAT1, αTAT1-S237A, TAK1, and TAB1