Figure 7. Inactive TBK1 has a higher binding affinity for ACSL1.

(A) IP analysis with overexpressed TBK1 WT or TBK1 K³⁸A and endogenous ACSL1 of primary hepatocytes. (B) IP analysis with overexpressed ACSL1 and TBK1 (WT, K³⁸A, S¹⁷²A) in HEK293 cell line. (C) IP analysis with overexpressed ACSL1 and each domain deleted TBK1 mutants (Full length, ΔULD, and ΔC-terminal). (D) The level of GST and pTBK1 (Ser¹⁷²) in each purified GST-TBK1 from sf9 cells for in vitro binding assay. (E) In vitro binding assay with full length TBK1 (WT), C-terminal truncated TBK1 (ΔC WT), C-terminal truncated TBK1 with K³⁸A mutation (ΔC K³⁸A), and C-terminal truncated TBK1 with S¹⁷²A mutation (ΔC S¹⁷²A). n = 3 replicates per group. (F) Quantified level of GST normalized by flag (ACSL1). *p < 0.05 (Student t-test). (G) Cayman 10576 (20 μM, pre-treatment for 30 min and 3 hours of fatty acid oxidation assay) increases fatty acid oxidation in primary hepatocytes. n = 6 replicates per group. **p < 0.01 (Student t test). (H) Fatty acid oxidation rate in TBK1 WT or TBK1 K³⁸A overexpressed hepatocytes from LTKO mice. n = 6 replicates per group. *p < 0.05, (two-way ANOVA followed Holm-Sidak’s multiple comparisons test). All IP analysis were replicated in more than three experiments. All data in the figure are shown as the mean ± SEM. See also Figure S6C and S7.