2.5. In Vitro Phosphatase Assay

PPM1A human recombinant and active ERK1 were purchased from Prospec (Ness-Ziona, Israel) and SignalChem (Richmond, British Columbia, Canada), respectively. Glutathione S-transferase (GST)-fused PPARγ was incubated with active ERK1 in kinase assay buffer (25 mmol/L Tris-HCl, pH 7.5, 5 mmol/L β-glycerophosphate, 2 mmol/L DTT, 0.1mmol/L Na₃VO₄, 10 mmol/L MgCl₂) containing 20 μmol/L ATP for 20 min at 30 °C. Then, purified phosphorylated GST-fused PPARγ was added into PPM1A human recombinant containing PPM1A phosphatase buffer [38] and incubated 30 min at 30 °C. To check the effect of catalytic inactive mutants of PPM1A (R174G and D239N) in dephosphorylation of PPARγ, HEK-293 cells were transfected with mutants expressing vector. Immunopurified PPM1As were incubated with phosphorylated GST-fused PPARγ in PPM1A phosphatase buffer for 30 min at 30 °C. All reactions were terminated by the addition of protein sample buffer. Phosphorylation of PPARγ was analyzed by SDS-PAGE with phospho-specific antibody against PPARγ at Ser273, anti-GST, or PPM1A antibodies.