<p>A-B) The positional scanning peptide library technology was used to determine the optimal phosphorylation motif for recombinant A) GST-TBK1 WT or B) kinase-dead GST-TBK1 K38A as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041494#pone.0041494-Hutti3" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041494#pone.0041494-Turk1" target="_blank">[28]</a>. Briefly, 198 peptide libraries were phosphorylated in individual kinase assays. The sequence for these libraries is Y-A-X-X-X-Z-X-S/T-X-X-X-X-A-G-K-K-biotin (Z = fixed amino acid, X = equimolar mixture of amino acids excluding Ser, Thr, and Cys). After binding to a streptavidin-coated membrane, phosphorylation was visualized by the incorporation of <sup>32</sup>P. C) Primary and secondary selections for TBK1, as determined in A). D) 50 µM of the indicated peptide was phosphorylated in an <i>in vitro</i> kinase assay with recombinant GST-TBK1 for 30 min. Phosphorylation of each peptide is shown as a percentage of the rate of phosphorylation of TBK1-Tide, the optimal peptide substrate for TBK1. Error bars are standard deviation.</p
