Fig. 2.

Effects of TBK1 ALS mutations on protein–protein interactions. (A) Impact of TBK1 ALS mutations on its dimerization. Expression constructs of Flag-tagged various TBK1 mutations from ALS patients were cotransfected with HA-tagged WT TBK1 into TBK1 knockout 293T cells, and cell lysates were prepared 24 h later and precipitated with anti-Flag antibodies (M2). The interaction between 2 differentially tagged TBK1 was assayed by western blot with anti-Flag and HA antibodies of the IP samples. (B) Reduction of TBK1 protein in cells derived from patients carrying G217R mutation. Control and patient fibroblasts were either untreated or treated with TBK1 inhibitor MRT67307 for 3 h, and total protein lysates were prepared for western blot analysis of TBK1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-Actin expression with specific antibodies. Lysates prepared from HeLa cells were also included as a control. (C) Coexpression of TBK1 mutants with a kinase-dead TBK1 improves the stability of mutant TBK1. HA-tagged G217R, M559R TBK1, and WT control were cotransfected with Flag-tagged WT TBK1 or D135N TBK1 into 293T cells; 24 h later, cell lysates were prepared for IP with anti-Flag beads. The expression and interaction of TBK1 mutants were analyzed by western blots with antibodies against HA, Flag, and TBK1.