2.8. In vitro Kinase Assay

In vitro kinase assay was performed as previously described [41]. Briefly, 1 μg of the purified PPARγ LBD was incubated with butyrolactone I, pioglitazone, and roscovitine at various concentrations ranging from 0.08 to 10 μM in the assay buffer (25 mM Tris-HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na₃VO₄, 10 mM MgCl₂) containing 25 μM ATP for 30 min at 30 °C. Then, active CDK5/p35 (Eurofins Scientific, Dundee, UK, Cat. No. 14-477) was added and incubated with PPARγ LBD for 30 min more at 30 °C. The phosphorylated PPARγ LBD by CDK5/p35 was detected by western blotting using an anti-CDK substrate antibody targeting phospho-Ser in a [K/R]-S-P-X-[K/R] motif (Cell Signaling Technology, Danvers, MA, USA, Cat. No. 9477) and an anti-PPARγ antibody targeting residues surrounding His466 of PPARγ (Cell Signaling Technology, Cat. No. 2443).