ATP K_m determination for TBK1 and IKKε.

<p>Enzymatic reactions of A) TBK1 or B) IKKε were incubated at room temperature with 10 ATP concentrations varying from 333 µM to 0.017 µM in three fold dilutions. Reactions were sampled on the Caliper EZReader system at 9.35 minute intervals over a 3 hour period. Percent conversions were calculated from relative heights of product and substrate peaks and used to calculate velocity and ATP K_m in Graph Pad Prism.</p

Activity comparison for TBK1 and IKKε.

<p>Number of active compounds (N) detected in each screen for total number detected (unfiltered), number after drug like filtering (filtered.drug-like), hits from the LOPAC and Kinase libraries, and the number of chemical clusters and singleton hits as described in the text.</p

Identification of the optimal phosphorylation motif for TBK1.

<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 ³²P. 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

Distribution of compound activity.

<p>A–B) The kinase library was screened at 10 µM in a single concentration format against enzymatic reactions of A) TBK1 and B) IKKε. The distribution of activity is shown as a frequency histogram based on the number of compounds active at each level (% Inhibition). The data follow a normal distribution.</p

Comparison of unfiltered and filtered <i>M. smegmatis</i>.

<p>Unfiltered <i>M. smegmatis</i> under 40x magnification (A) and plated onto agar (B); <i>M. smegmatis</i> filtered through 5-µm pore filter under 40 x magnification (C) and plated onto agar (D), scale bar applies to both A and C.</p

Recovery of <i>M. smegmatis</i> after Vortexing and Filtration.

<p>Mean ±Standard Deviation; n = 4.</p

Histogram of unfiltered, vortexed and filtered <i>M. smegmatis</i>.

<p>Distributions of resorufin fluorescence signals from 384 wells of a 384-well plate contained unfiltered (A), vortexed (B) or filtered (C) <i>M. smegmatis</i>. After the treatment, the bacteria were distributed into the 384-well plates followed by the addition of resazurin, which was converted to resorufin by the living bacteria.</p

Agreement analysis of duplicate plates from unfiltered, vortexed and filtered bacteria.

<p>Correlation of two duplicate assay plates tested against LOPAC compounds using unfiltered bacteria (A), vortexed bacteria (B) and filtered bacteria (C).</p

Supplemental_Material_for_Hull-Ryde_et_al – Supplemental material for Identification of Cosalane as an Inhibitor of Human and Murine CC–Chemokine Receptor 7 Signaling via a High-Throughput Screen

<p>Supplemental material, Supplemental_Material_for_Hull-Ryde_et_al for Identification of Cosalane as an Inhibitor of Human and Murine CC–Chemokine Receptor 7 Signaling via a High-Throughput Screen by Emily A. Hull-Ryde, Melissa A. Porter, Kenneth A. Fowler, Dmitri Kireev, Kelin Li, Catherine D. Simpson, Maria F. Sassano, Mark J. Suto, Kenneth H. Pearce, William Janzen and James M. Coghill in SLAS Discovery</p