Fig 2: Serine/threonine-protein kinase TBK1 antibody screening by immunofluorescence.U2OS WT and TBK1 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated TBK1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with yellow and magenta dashed line, respectively. Antibodies were tested at 1.0 µg/ml. When the concentration was not indicated by the supplier, we tested the antibodies at 1/500 or 1/1000. At this concentration, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: NB100-56705 at 1/1000; 28397-1-AP at 1/500; 67211-1-Ig at 1/1000; PA5-17478 at 1/1000; 703154 at 1/500; ab12116 at 1/1000; ab40676 at 1/1500; ab109735 at 1/500; GTX113057 at 1/700, 38066 at 1/500, 3504 at 1/500. Bars = 10 µm.

Fig 3: 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.

Fig 4: Influence of R563G TBK1 mutation on autophagy flux and expression levels of pro-inflammatory cytokines. (A) Lymphoblasts from pooled control cell lines and from the E7 patient were seeded at an initial density of 1 × 106 cells × mL-1 in RPMI containing 10% FBS for 24 h. Hydroxychloroquine (HCQ) was added during the last 3 h when indicated. Each point represents the mean of four independent experiments. (B) Quantitative real-time PCR determination of messenger RNA levels of pro-inflammatory cytokines IL-1, IL-6, and TNF-a, normalized by ß-Actin messenger RNA levels. The data represent means ± SEM of four independent experiments. * p < 0.05, significantly different from control cells.

Fig 5: Effects of R563G TBK1 mutation on cell viability and ROS generation. (A) Lymphoblasts from pooled control cells and from E7 ALS patient were seeded at an initial density of 1 × 106 cells × mL-1 in the absence or presence of 10% FBS for 24 h. Cell viability was assessed by the MTT assay. The data represent the means ± SEM of four independent experiments carried out in triplicate. (B) Cells were incubated in RPMI medium containing 10% FBS for 24 h. Subsequently, about 200,000 cells were incubated with the CM-H2DCFDA probe for 30 min. The data show the means ± SEM of four independent experiments. * p < 0.05, *** p < 0.001, significantly different from control cells.