P81 filters

For dot blot assays, 30 μg of aliquots of each of nonmethylated K209 peptide or phosphorylated T210 peptide of Plk1 was incubated with roughly 0.5 μg of methyltransferase moieties immunoprecipitated from 293T cells in the presence of 0.1 mM S-adenosyl-methionine (SAM; Sigma) at 30°C for 1 hour in the buffer [50 mM tris-HCl (pH 8.0), 10% glycerol, 20 mM KCl, 5 mM MgCl₂, and 1 mM phenylmethyl sulfonyl fluoride]. Reactions were spotted onto nitrocellulose membranes and immunoblotted with an α-Plk1 K209me1 antibody. The monomethylated Plk1 K209 peptide served as a control.
For protein methylation assays, 1-μg amount of bacterially or insect cell–expressed recombinant wild type or K209R GST–N-Plk1 was incubated overnight with G9a-SET fragments immunoprecipitated from 293T cells in the presence of 2 μCi of ³H-labeled SAM at 30°C. Reactions were spotted onto P81 filters (Whatman) and washed four times with 50 mM NaHCO₃ (pH 9.0) before scintillation counting. Alternatively, reactions were quenched by adding an equal volume of 2× SDS sample buffer. Radiolabeled protein was separated by SDS-PAGE gel, followed by Coomassie blue staining or autoradiography.

Myocardial protein kinase G (PKG) activity was assessed in homogenized tissue samples. Tissues samples were homogenized in 25 mmol/l Tris (pH 7.4), 1 mmol/l EDTA, 2 mmol/l EGTA, 5 mmol/l dithiothreitol (DTT), 0.05% Triton X‐100, and protease inhibitor cocktail (Sigma Aldrich) and centrifuged for 5 minutes. Supernatants containing equal amounts of total protein were analyzed for PKG activity. The reaction mixture contained (at final concentration): 40 mmol/l Tris‐HCl (pH·7.4), 20 mmol/l magnesium acetate, 0.2 mmol/l [32P] ATP (500–1,000 c.p.m.·pmol⁻¹) (GE Healthcare LifeScience, Little Chalfont, U.K.), 113 mg/ml heptapeptide (RKRSRAE), 3 mmol/l cGMP (Promega, Madison, WI) and a highly specific inhibitor of cAMP‐dependent protein kinase (5–24, Merck Millipore). The reaction mixtures were incubated at 30°C for 10 minutes, followed by termination of the reaction by spotting 70·μl of the reaction mix onto Whatman P‐81 filters, which were then soaked with 75·mmol/l H₃PO₄ for 5 minutes and washed three times with 75·mmol/l H₃PO₄ to remove any unbound [32P] ATP. Filters were rinsed with 100% ethanol and air dried before quantification. For quantification of PKG activity, counts were taken in a Wallac 1409 Liquid Scintillation Counter using universal scintillation cocktail. Specific activity of PKG was expressed as pmol of [³²P] incorporated into the substrate (pmol/minute/mg protein).