PPARγ Phosphorylation Assay

Immuno-purified WT or S273A mutant of PPARγ were incubated with active cdk kinases in kinase assay buffer containing ATP for 15 min at 30°C. Positive controls, either purified histone H1 (Millipore) or Rb (Cell Signaling Technology) were used. Several PPARγ ligands were pre-incubated with substrates for 30 min before the assay was performed.

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Choi J.H., Banks A.S., Estall J.L., Kajimura S., Bostrom P., Laznik D., Ruas J.L., Chalmers M.J., Kamenecka T.M., Bluher M., Griffin P.R, & Spiegelman B.M. (2010). Obesity-linked phosphorylation of PPARγ by cdk5 is a direct target of the anti-diabetic PPARγ ligands. Nature, 466(7305), 451-456.

Publication 2010

Assay Atp kinase Buffer Histone h1 Kinases Ligands Pparγ

Corresponding Organization : Harvard University

Other organizations : Scripps Research Institute, Leipzig University

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Protocol cited in 11 other protocols

Variable analysis

independent variables

Purified WT or S273A mutant of PPARγ
Several PPARγ ligands

dependent variables

Phosphorylation of PPARγ

control variables

Kinase assay buffer containing ATP
Incubation time (15 min at 30°C)

controls

Positive controls: Purified histone H1 or Rb

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