Supplementary Figure 6: Functional analysis of TBK1 missense variance

(a) Co-immunoprecipitation (Co-IP): Flag-TBK1-WT or the indicated mutants were expressed in HEK293T cells. The fusion proteins were purified and immobilized from HEK293T cell lysates using anti-Flag beads followed by incubation with recombinant GST-OPTN. Co-precipitated GST-OPTN was detected with an antibody against OPTN (top panel). Protein levels of immobilized Flag-TBK1 WT were analyzed by immunoblotting (anti-Flag; lower panel). Flag-beads only (IgG only) were used as a negative control. WT (wild-type); KD (kinase-dead TBK1 mutation; p.Lys38Ala). (b) GST pull-down assay (upper panel): Flag-TBK1-WT or the indicated mutants from HEK293T cell lysates (as in a) were incubated with recombinant, immobilized GST-OPTN. Equal GST-OPTN protein levels were confirmed by Ponceau S staining. Arrowhead points to full-length GST-OPTN. Flag-TBK1 protein expression was analyzed by immunoblotting (lower panel). (c) Co-IP of myc-IRF3 and Flag-TBK1-WT or the indicated mutants from HEK293T cells using anti-Flag agarose. (Co-)Immunoprecipitated proteins were analyzed by IB with the indicated antibodies (upper panel). Asterisk points to heavy IgG bands. Flag-TBK1 and myc-IRF3 protein expression was analyzed by IB with the indicated antibodies (lower panel). IB images are representatives of three repetitions.