Fig 2: Quantification of mRNA and protein expression level of TBK1 in patient-derived fibroblasts. (a) Quantitative RT-PCR analysis of TBK1 mRNA level in fibroblasts from healthy control and the patient with p.Ile334Thr mutation. Values are expressed as mean ± SEM. ***p < 0.0001; unpaired t test. (b) Western blot analysis of TBK1 and phosphorylated IRF3 protein expression level in patient-derived fibroblasts. ß-actin was used as a loading control. (c) Quantification of TBK1 level. Values are expressed as mean ± SEM. *p < 0.05; unpaired t test

Fig 3: Detection of p.Ile334Thr in TBK1 (NP_037386.1). (a) Sanger sequencing showed the heterozygous of the mutation. (b) The overview of protein. The protein is colored gray, and the side chain of the mutant residue is colored magenta. (c) Close-up of the mutation. The protein is colored gray, and both the wild-type (green) and mutant (red) residue are shown. (d) Conservation among multiple species at position 334

Fig 4: Serine/thronine-protein kinase TBK1 screening by immunoprecipitation.U2OS lysates were prepared and IP was performed using 1.0 µg of the indicated TBK1 antibodies pre-coupled to either protein G or protein A Sepharose beads. Samples were washed and processed for immunoblot with the indicated TBK1 antibody. For immunoblot, ab40676, ab12116 and 67211-1-Ig were used. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitate.

Fig 5: Serine/threonine-protein kinase TBK1 antibody screening by immunoblot.Lysates of U2OS (WT and TBK1 KO) were prepared and 50 µg of protein were processed for immunoblot with the indicated TBK1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier, except for antibodies 28397-1-AP, PA5-17478, 703154, ab40676, and ab109735, which were titrated at 1/10000 because the signal was too strong following the supplier’s recommendations. Antibody dilution used: NB100-56705 at 1/500; 28397-1-AP at 1/10000; 67211-1-Ig at 1/1000; PA5-17478 at 1/10000; 703154 at 1/10000; ab12116 at 1/600; ab40676 at 1/10000; ab109735 at 1/10000; GTX113057 at 1/2000, 38066 at 1/1000, 3504 at 1/1000. Predicted band size: ~83 kDa.