Figure 7: Chiglitazar differentially affects cofactor recruitment upon CDK5-mediated phosphorylation. AlphaScreen assay was performed to evaluate the recruitment of six representative LXXLL peptides to PPARγ-LBD following treatment with 2 μM of Chi, Ros, and Pio as described in Materials and Methods. The S273 phosphorylation status of PPARγ-LBD was varied by adding different components to the in vitro phosphorylation reaction as indicated. For instance, “LBD + CDK5 − ATP + agonist” denotes no phosphorylation completed by active CDK5/p25 without ATP in the reaction (indicated as black bar). “LBD + CDK5 + ATP + agonist” indicates that the phosphorylation was partially repressed in the presence of agonist (indicated as grey bar). Finally, “(LBD + CDK5 + ATP) + agonist” signifies that the phosphorylation was completed before the addition of agonist to the reaction (indicated as light grey bar). All reactions were then directly applied to the AlphaScreen assay following a conventional procedure as described in Materials and Methods. The baseline for cofactor recruitment, independent of ligand binding, was calculated using RLUs from reactions performed in the presence of cofactor peptides divided by control reactions lacking peptide (background signal). Induction of cofactor recruitment by agonist is represented in fold change using RLUs from the cofactor binding reaction with agonist divided by the vehicle control sample (0.1% (vol/vol) DMSO).