P81 phosphocellulose paper

Batch reactions (150 μL final volume) contained 15 μg/mL receptor, 10 mM MgCl₂, 0.5 mM ATP, 1–6 μCi [γ-32P]-ATP (PerkinElmer, 10 mCi/mL, 25–50 cpm/pmol) and synthetic peptide KKEEEEYMMMMG at concentrations of 0.05 mM–2 mM in a Buffer B. Some reactions contained insulin or IGF1 at concentrations of 200 nM. Reactions were stopped at various times by removing 25 μL from the batch reaction and quenched by adding to 45 μL cold 10% (w/v) trichloroacetic acid. Samples were centrifuged at 10,000 × g for 2 min and 35 μL of supernatant was spotted on P81 phospho-cellulose paper (Whatman). Excess radiolabeled ATP was removed by three 10 min washes in cold 0.5% phosphoric acid followed by a final wash in acetone. The P81 paper circles were dried and activity in counts per min (cpm) was measured in a scintillation counter.

CaMKII activity was measured in ventricular homogenate using Syntide-2, a synthetic CaMKII-specific substrate peptide. Hearts were isolated and ventricles homogenized in lysis buffer (50 mmol/L HEPES, 10% ethylene glycol, 2 mg/ml BSA, 5 mmol/L EDTA, pH 7.5), and assayed immediately without freezing. The assay buffer contained 50 mmol/L HEPES, 10 mmol/L magnesium acetate, 1 mg/ml BSA, 20 µmol/L Syntide-2, 1 mmol/L DTT, 400 nmol/L [γ-³²P]ATP, pH 7.5 and either 1 mmol/L EGTA (for autonomous activity) or 500 µmol/L CaCl₂, plus 1 µmol/L calmodulin (for maximal activity). The reaction was carried out at 30 °C for 10 min and blotted onto Whatman P81 phosphocellulose paper. Percent activation was calculated as the ratio of autonomous to maximal activity for each sample.

HAT activity was determined using a filter-binding assay as previously described, with some modifications (Han et al., 2007a (link); Tanner et al., 1999 (link)). Samples were incubated at 30°C for 30 minutes in a 30 μl reaction mixture containing 50 mM Tris-HCl, pH 8.0, 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 1 mM DTT, 10 mM sodium butyrate, 250 mM NaCl, 1 mM PMSF, 1.6 μM [³H]acetyl-CoA (8.6 Ci/mmol, PerkinElmer), and recombinant WT or mutant forms of AfRtt109, AfAsf1 and histones H3 and H4, as specified. 7.5 μl of each reaction were spotted onto P-81 phosphocellulose paper (Whatman), air dried, and washed five times with 50 ml of buffer containing 50 mM NaHCO₃ at pH 9.0, and once with 50 ml of acetone. The amount of radioactivity of each air-dried filter paper was measured using a liquid scintillation counter. To visualize the amount of each protein and detect acetylated ones, each reaction mixture was resolved by 15% SDS-PAGE and the gels were either stained with SYPRO Ruby (BioRad) or dried and exposed to photo films after incubation with Amplify (Amersham) for 30 minutes. To detect H3K56 acetylation, HAT assays were performed as before but using unlabeled acetyl-CoA and analyzed by Western blot using antibodies recognizing acetylated H3K56.