Figure S1 - Protein Kinase C-Dependent Dephosphorylation of Tyrosine Hydroxylase Requires the B56δ Heterotrimeric Form of Protein Phosphatase 2A
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PKC-mediated phosphorylation of PP2A with B56δ subunit. N2a cells were transfected either with FLAG-B56δ S566A mutant or FLAG- B56δ wt then the lysates were immunoprecipitated using anti-FLAG antibody. Immunoprecipitated PP2A holoenzymes were eluted by adding FLAG peptide (100 µg/ml, Sigma-Aldrich, St. Louis, MO) for 10 min. Eluted PP2A with B56δ subunit complex was incubated with purified PKC and [γ-32P] ATP without or with PKC activator for 5 min. Immune-complexes were separated with 4-20% SDS-PAGE, and phosphorylation of PP2A subunits measured using autoradiography. After autoradiography, bands corresponding to each subunit (A, B, and C) were excised and 32P incorporation measured using a scintillation counter (cpm). The bar graph represents the cpm as means ± s.e.m. (n = 3). *, P (EPS)
