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. 2023 Sep 1;55(10):2162–2176. doi: 10.1038/s12276-023-01060-7

Fig. 3. TKT phosphorylation at Thr287 is critical for TKT activation, cytoplasmic localization, and TKT-E3 ligase FBXL6 interaction.

Fig. 3

a Examination of the TKT protein sequence identified Thr287 in TKT as evolutionarily conserved in different species. b FBXL6 binds to wild-type TKT but not its mutant (T287A). Huh7 cells were transfected with the indicated plasmids for 48 h, followed by pulldown with anti-Flag IgG. c–i Huh7 and Hepa1-6 cells were transfected with the wild-type TKT or its TKT T287A mutant plasmid for 48 h. A portion of the cells was harvested for Western blotting (c). Another portion was used for staining TKT and its mutant (T287A) (d, e), or detection of TKT activity using a TKT activity assay kit (f). Other portions were replated into 96-well plates for a cell proliferation assay (g) or into Transwell plates and incubated overnight for a migration assay (h, i). The nuclear and cytoplasmic fluorescence intensity in the cells described in (d) was determined by ImageJ software. Scale bar, 20 μm. Unpaired t test was used in (e); one-way ANOVA with Tukey’s multiple comparisons test was used in (f, i). **p ≤ 0.01, ***p ≤ 0.001.