Experimentally measured PLCγ1activation kinetics in DP thymocytes stimulated with TCR ligands of different affinities and robustness of <i>in silico</i> models.


<p>(A) Immunoblots showing Y<sub>783</sub>-phosphorylated (upper panels) and total (lower panels) PLCγ1 protein amounts in <i>RAG2<sup>−/−</sup>MHC<sup>−/−</sup> OT1 TCR-transgenic</i> DP thymocytes stimulated for the indicated times with MHCI tetramers presenting the indicated altered peptide ligands (APL). (B) Phospho-PLCγ1 levels normalized to total PLCγ1 protein amounts plotted over time for the indicated APLs. Their TCR affinity decreases in the order OVA (black)>Q4R7 (red)>Q4H7 (blue)>G4 (green). Band intensities were quantified via scanning and analysis with <i>ImageJ</i> software. Representative of several independent experiments. (C) Variation of the Kulback-Leibler distance D<sub>KL</sub> with <i>R</i> for models M1–M3 (blue, red and black, respectively), M7 (yellow), and M4–M6 (orange, purple, and maroon, respectively) at high initial Itk (Itk<sup>0</sup> = 140 molecules) and PIP<sub>3</sub> concentrations (PIP<sub>3</sub><sup>0</sup> = 530 molecules), representing high-affinity OVA stimulation for <i>τ</i><sub>p</sub> = 2 min and <i>A</i> (shown as <i>A</i><sub>avg</sub>) = 40 molecules. Note we use <i>A</i> to represent the amplitude <i>A</i><sup>expt</sup> in experiments measuring fold change in Itk phosphorylation (see the main text for further details). The vertical orange bar indicates R<i><sup>expt</sup></i> for OVA. Color legend in (D). (D) The color map shows which model is most robust (has the lowest D<sub>KL</sub>) as <i>R<sup>expt</sup></i> and <i>A</i> (shown as <i>A</i><sub>avg</sub>) are varied for the same parameters as in (C). The color legend is depicted on the right.</p

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