Fig. 3.
TBK1 R357Q is a monomeric and functional kinase. (A) Dissociation constant (Kd) of selected TBK1 mutants (residues 1 to 657) determined by AUC experiments. (B) Gel filtration chromatograms of the indicated recombinant TBK1 mutants showing milli-absorbance units (mAU) over elution volume. Positions of molecular mass standards were also marked. TBK1 (1 to 657) mutants were expressed in insect cells and purified. These ALS-related mutants were all in the kinase-dead D135N background. Recombinant proteins were injected onto a Superdex 200 Increase 10/300GL column (GE Healthcare) at a flow rate of 0.5 mL/min in a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, and 1 mM tris(2-carboxyethyl)phosphine (TCEP), and the ultraviolet absorbance (mAU) at 280 nm was recorded. (C) TBK1 R357Q can phosphorylate a different TBK1 molecule when coexpressed. GST-tagged WT TBK1 (Left) or D135N TBK1 (Right) was coexpressed with Flag-tagged WT, D135N, S172A, R357Q, E696K TBK1, or EGFP as a control in TBK1 knockout 293T cells; 24 h after transfection, cell lysates were prepared, and anti-Flag beads were used to IP Flag-tagged proteins. The profiles of pS172 TBK1 and total TBK1 from different samples were determined by western blots with respective antibodies.